cyclic-gmp and Hypoxia

Nitric oxide (NO) plays a role in a wide range of physiological processes. Aside from its widely studied function in the regulation of vascular function, NO has been shown to impact steroidogenesis in a number of different tissues. The goal of this review is to explore the effects of NO on steroid production and further, to discern its source(s) and mechanism of action. Attention will be given to the regulation of NO synthases in specific endocrine tissues including ovaries, testes, and adrenal glands. The effects of hypoxia on generation of NO and subsequent effects on steroid biosynthesis will also be examined. Finally, a potential model for the interaction of hypoxia on NO synthesis and steroid production is proposed.

Topics: Adrenal Glands; Animals; Cyclic GMP; Cysteine; Enzyme Activation; Female; Guanylate Cyclase; Heme; Humans; Hypoxia; Male; Models, Biological; Nitric Oxide; Nitric Oxide Synthase Type III; Ovary; Pregnancy; Protein Processing, Post-Translational; Steroids; Testis; Tyrosine

Pulmonary vascular responses elicited by hypoxia and NO-cGMP signaling are potentially influenced by ROS and redox mechanisms that change during the progression of disease processes. Our studies in endothelium-rubbed bovine pulmonary arteries suggest increased glucose-6-phosphate dehydrogenase levels (compared to coronary arteries) seem to maintain a tonic peroxide-mediated relaxation removed by hypoxia through NADPH fueling superoxide generation from Nox oxidase. The activities of glucose-6-phosphate dehydrogenase, oxidases (i.e., Nox4), and systems metabolizing superoxide and peroxide markedly influence hypoxic pulmonary vasoconstriction (HPV). Activation of soluble guanylate cyclase and cGMP protein kinase seems to participate in peroxide-elicited relaxation. Endogenous NO helps maintain low pulmonary arterial pressure and suppresses HPV. Multiple redox processes potentially occurring during the progression of pulmonary hypertension may also attenuate NO-mediated relaxation beyond its scavenging by superoxide, including oxidation of guanylate cyclase heme and thiols normally maintained by cytosolic NADPH redox control.

Topics: Animals; Cyclic GMP; Humans; Hypoxia; Nitric Oxide; Oxidation-Reduction; Pulmonary Artery; Signal Transduction

Mitochondria, the site of oxidative energy metabolism in eukariotic cells, are a highly organised structure endowed with different enzymes and reactions localized in discrete membranes and aqueous compartments. Mitochondrial function is regulated in complex ways by several agonists and environmental conditions, through activation of specific transcription factors and signalling pathways. A key player in this scenario is nitric oxide (NO). Its binding to cytochrome c oxidase in the mitochondrial respiratory chain, which is reversible and in competition with oxygen, plays a role in acute oxygen sensing and in the cell response to hypoxia. Evidence of the last two years showed that NO has also long-term effects, leading to biogenesis of functionally active mitochondria, that complement its oxygen sensing function. Mitochondrial biogenesis is triggered by NO through activation of guanylate cyclase and generation of cyclic GMP, and yields formation of functionally active mitochondria. Thus, the combined action of NO at its two known intracellular receptors, cytochrome c oxidase and guanylate cyclase, appears to play a role in coupling energy generation with energy demand. This may explain why dysregulation of the NO signalling pathway is often associated with the pathogenesis of metabolic disorders.

Topics: Animals; Cyclic GMP; Electron Transport Complex IV; Guanylate Cyclase; Humans; Hypoxia; Mice; Mice, Transgenic; Mitochondria; Models, Biological; Nitric Oxide; Nitric Oxide Synthase Type III; Oxygen; Signal Transduction; Transcription, Genetic

Topics: Asphyxia Neonatorum; Biomarkers; Blood Circulation; Cyclic GMP; Female; Fetal Diseases; Fetal Growth Retardation; Guanylate Cyclase; Humans; Hypoxia; Infant, Newborn; Natriuretic Peptides; Pre-Eclampsia; Pregnancy; Receptors, Atrial Natriuretic Factor; Uterine Contraction; Uterus

At birth, a marked decrease in pulmonary vascular resistance allows the lung to establish gas exchange. Persistent pulmonary hypertension of the newborn (PPHN) occurs when this normal adaptation of gas exchange does not occur. We review animal models used to study the pathogenesis and treatment of PPHN. Both acute models, such as acute hypoxia and infusion of vasoconstrictors, and chronic models of PPHN created both before and immediately after birth are described. Inhaled nitric oxide is an important emerging therapy for PPHN. We review nitric oxide receptor mechanisms, including soluble guanylate cyclase, which produces cGMP when stimulated by nitric oxide, and phosphodiesterases, which control the intensity and duration of cGMP signal transduction. A better understanding of these mechanisms of regulation of vascular tone may lead to safer use of nitric oxide and improved clinical outcomes.

Topics: Animals; Constriction, Pathologic; Cyclic GMP; Disease Models, Animal; Humans; Hypertension, Pulmonary; Hypoxia; Infant, Newborn; Ligation; Nitric Oxide Synthase; Persistent Fetal Circulation Syndrome; Pulmonary Artery; Vasodilation

Topics: Adenosine; Animals; Calcimycin; Carbon Dioxide; Cerebrovascular Circulation; Cyclic GMP; Endothelium, Vascular; Fetus; Humans; Hydrogen-Ion Concentration; Hypoxia; Infant, Newborn; Oxygen; Penicillamine; Prostaglandins; S-Nitroso-N-Acetylpenicillamine; Sheep; Vascular Resistance

Anoxia has been compared with ischaemia. The abrupt restoration of either oxygen of flow may accelerate cardiac damage. Anoxic stimulation of glycolysis (Pasteur effect) is inhibited during ischaemia by lactate and proton accumulation at the levels of phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase. Anaerobic glycolysis provides lactate and ATP; breakdown of the latter provides protons. During partial respiration thought to occur in partial ischaemia, continued production of CO2 is a factor contributing to intracellular acidosis; mitochondrial ATP when formed by continued respiration also yields protons when ultimately broken down. The endoproducts of aerobic glycolysis (pyruvate and NADH) are transported into the mitochondria by the malate-aspartate cycle and by pyruvate dehydrogenase activity. Adenine nucleotide transferase activity normally transfers the mitochondrially-made ATP to the cytoplasm, but acyl CoA accumulates in ischaemia (or during perfusions with high circulating free fatty acids) to inhibit the transferase. The mitochondrial creatine kinase is thought to transform ATP transported outwards into creatine phosphate which can permeate the outer mitochondrial membrane. Further compartmentation of ATP may be by other creatine kinase isoenzymes or in relation to the cell membrane. The glycogenolytic-sarcoplasmic reticulum complex links a glycogen pool to the sarcoplasmic reticulum. Cyclic AMP may regulate admission of calcium to the cell during the plateau of the action potential and promote calcium uptake by the sarcoplasmic reticulum by phosphorylation of phospholamban. The latter promotes the activity of the calcium-transport ATPase. Calcium and cyclic AMP may also interact at the level of the contractile proteins where cyclic AMP phosphrylates troponin. Cyclic GMP generally has opposite effects to cyclic AMP and undergoes opposite changes in the frog cardiac cycle to those of cyclic AMP. A present it is reasonable to suppose that physiological effects of adrenaline or of cholinergic agents on the myocardium are mediated by cyclic AMP or cyclic GMP, respectively, but this hypothesis still lacks firm support. There is an association between tissue cyclic AMP and ventricular fibrillation after coronary ligation, and direct evidence for a role of cyclic AMP in promoting arrhythmias has been obtained by studies on the ventricular fibrillation threshold in the rat heart. However, there are other mechanisms, involving first the ef

Topics: Adenosine Triphosphate; Animals; Arrhythmias, Cardiac; Calcium; Cyclic AMP; Cyclic GMP; Energy Metabolism; Fatty Acids, Nonesterified; Glucose; Glycolysis; Humans; Hydrogen-Ion Concentration; Hypoxia; Lactates; Mitochondria, Heart; Myocardial Infarction; Myocardium; Oxygen Consumption; Pyruvate Kinase; Pyruvates; Rats

Topics: Airway Obstruction; Asthma; Autonomic Nervous System; Cyclic AMP; Cyclic GMP; Epinephrine; Humans; Hypoxia; Muscle, Smooth; Parasympathomimetics; Receptors, Adrenergic

A regulatory role for adenosine 3',5'-monophosphate (cyclic AMP) in the production of the renal hormone rythropoietin following erythropoietic stimulation with cobaltous chloride hexahydrate is proposed. Studies in rates reveal a temporal relationship between renal cyclic AMP levels and plasma titers of erythropoietin. In addition, cobalt increases the activity of an erythropoietin-generating enzyme (renal erythropoietic factor) with maximal enzyme activity occurring after the rise in cyclic AMP levels but before the increase in erythropoietin titers. This increase in renal cyclic AMP is localized to the renal cortex. Cobalt stimulates renal cortical adenylate cyclase but has no effect on renal cyclic nucleotide phosphodiesterase. The addition of cyclic AMP (3 time 10-6 M) and a partially purified cyclic AMP-dependent protein kinase from rat kidney to an inactive preparation of renal erythropoietic factor increases the ability of renal erythropoietic factor to generate erythropoietin. Data from the polycythemic mouse assay, a bioassay used to quantitate erythropoietic activity of test substances, indicate that dibutyryl cyclic AMP is erythropoietically active with respect to its ability to increase radioactive-labelled iron (59Fe) incorporation into heme of newly formed red blood cells. Theophylline, which by itself is erythropoietically inactive, potentiated the erythropoietic effect of cobalt in polycythemic mice. These results suggest that cyclic AMP plays a significant role in the renal production of erythropoietin following cobalt administration. It is postulated that cobalt stimulates renal cortical adenyoate cyclase, thus increasing renal cyclic AMP levels. Cyclic AMP then activates a protein kinase which subsequently stimulates renal erythropoietic factor to generate erythropoietin. A similar cyclic AMP mechanism may be operative after erythropoietic stimulation by exposure to hypoxia or prostaglandin treatment.

Topics: Adenine Nucleotides; Adenylyl Cyclases; Animals; Bucladesine; Cobalt; Cyclic AMP; Cyclic GMP; Enzyme Activation; Erythropoietin; Humans; Hypoxia; Kidney; Kidney Cortex; Mice; Phosphoric Diester Hydrolases; Prostaglandins; Protein Kinases; Rats; Stimulation, Chemical; Theophylline

Red blood cells (RBCs) mediate cardioprotection via nitric oxide-like bioactivity, but the signaling and the identity of any mediator released by the RBCs remains unknown. We investigated whether RBCs exposed to hypoxia release a cardioprotective mediator and explored the nature of this mediator. Perfusion of isolated hearts subjected to ischemia-reperfusion with extracellular supernatant from mouse RBCs exposed to hypoxia resulted in improved postischemic cardiac function and reduced infarct size. Hypoxia increased extracellular export of cyclic guanosine monophosphate (cGMP) from mouse RBCs, and exogenous cGMP mimicked the cardioprotection induced by the supernatant. The protection induced by hypoxic RBCs was dependent on RBC-soluble guanylate cyclase and cGMP transport and was sensitive to phosphodiesterase 5 and activated cardiomyocyte protein kinase G. Oral administration of nitrate to mice to increase nitric oxide bioactivity further enhanced the cardioprotective effect of hypoxic RBCs. In a placebo-controlled clinical trial, a clear cardioprotective, soluble guanylate cyclase-dependent effect was induced by RBCs collected from patients randomized to 5 weeks nitrate-rich diet. It is concluded that RBCs generate and export cGMP as a response to hypoxia, mediating cardioprotection via a paracrine effect. This effect can be further augmented by a simple dietary intervention, suggesting preventive and therapeutic opportunities in ischemic heart disease.

Topics: Animals; Cardiotonic Agents; Cyclic GMP; Erythrocytes; Humans; Hypoxia; Mice; Myocytes, Cardiac; Nitrates; Nitric Oxide; Rats; Soluble Guanylyl Cyclase

We sought to determine the influence of sildenafil on the diffusing capacity of the lungs for carbon monoxide (DLCO) and the components of DLCO (pulmonary capillary blood volume VC, and alveolar-capillary membrane conductance DM) at rest and following exercise with normoxia and hypoxia. This double-blind placebo-controlled, cross-over study included 14 healthy subjects (age = 33 +/- 11 years, ht = 181 +/- 8 cm, weight = 85 +/- 14 kg, BMI = 26 +/- 3 kg/m2, peak normoxic VO2 = 36 +/- 6 ml/kg, mean +/- SD). Subjects were randomized to placebo or 100 mg sildenafil 1 h prior to entering a hypoxic tent with an FiO2 of 12.5% for 90 min. DLCO, VC, and DM were assessed at rest, every 3 min during exercise, at peak exercise, and 10 and 30 min post exercise. Sildenafil attenuated the elevation in PAP at rest and during recovery with exposure to hypoxia, but pulmonary arterial pressure immediately post exercise was not different between sildenafil and placebo. Systemic 02 saturation and VO2peak did not differ between the two conditions. DLCO was not different between groups at any time point. VC was higher with exercise in the placebo group, and the difference in DM between sildenafil and placebo was significant only when corrected for changes in VC (DM/VC = 0.57 +/- 0.29 vs. 0.41 +/- 0.16, P = 0.04). These results suggest no effect of sildenafil on DLCO, but an improvement in DM when corrected for changes in VC during short-term hypoxic exposure with exercise.

Topics: Acute Disease; Adult; Capillaries; Cross-Over Studies; Cyclic GMP; Double-Blind Method; Endothelin-1; Exercise; Female; Heart Rate; Humans; Hypoxia; Male; Natriuretic Peptide, Brain; Oxygen; Piperazines; Pulmonary Alveoli; Pulmonary Circulation; Pulmonary Gas Exchange; Purines; Rest; Sildenafil Citrate; Stroke Volume; Sulfones; Vasodilator Agents

This study investigated the effect of the phosphodiesterase 5 inhibitor sildenafil on the pulmonary vascular response to hypoxia in humans and mice.. In a randomized, double-blind study, sildenafil 100 mg or placebo was given orally to 10 healthy volunteers 1 hour before breathing 11% O(2) for 30 minutes. Pulmonary artery pressure (PAP) was measured with an indwelling right heart catheter. The acute 56% increase in mean PAP produced by hypoxia during placebo treatment (mean PAP [mean+/-SD mm Hg]: normoxia 16.0+/-2.1 versus hypoxia 25.0+/-4.8) was almost abolished by sildenafil (normoxia 16.0+/-2.1 versus hypoxia 18.0+/-3.6), with no significant effect on systemic blood pressure. In the isolated perfused lung of wild-type and endothelial nitric oxide synthase (eNOS)-deficient mice, sildenafil markedly blunted acute hypoxic pulmonary vasoconstriction. Wild-type mice dosed orally with the drug (25 mg. kg(-1). d(-1)) throughout 3 weeks of exposure to hypoxia (10% O(2)) exhibited a significant reduction in right ventricular systolic pressure (placebo versus sildenafil: 43.3+/-9.9 versus 29.9+/-9.7 mm Hg, P<0.05) coupled with a small reduction in right ventricular hypertrophy and inhibition of pulmonary vascular remodeling. In eNOS mutant mice, sildenafil attenuated the increase in right ventricular systolic pressure but without a significant effect on right ventricular hypertrophy or vascular remodeling.. Sildenafil attenuates hypoxia-induced pulmonary hypertension in humans and mice and offers a novel approach to the treatment of this condition. The eNOS-NO-cGMP pathway contributes to the response to sildenafil, but other biochemical sources of cGMP also play a role. Sildenafil has beneficial pulmonary hemodynamic effects even when eNOS activity is impaired.

Topics: Adolescent; Adult; Animals; Cyclic GMP; Double-Blind Method; Genotype; Heart Ventricles; Humans; Hypertension, Pulmonary; Hypertrophy; Hypoxia; In Vitro Techniques; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Phosphodiesterase Inhibitors; Piperazines; Pulmonary Artery; Purines; Sildenafil Citrate; Sulfones; Ventricular Function, Right

The acute effects of the inhaled gas phase of cigarette smoke on pulmonary (PAP) and systemic (SAP) arterial pressures and on plasma arterial cGMP content were compared with those of inhaling 10, 20 and 80 ppm nitric oxide (NO) in one healthy adult volunteer spontaneously breathing a hypoxic gas mixture. Hypoxia (FIO2 0.12) induced a sustained, stable pulmonary vasoconstriction. Inhaled NO induced a dose-dependent fall in PAP; plasma cGMP rose from 39.4 (hypoxia) to 164 pmol/ml (hypoxia plus 80 ppm NO). Exposure to cigarette smoke induced a rapid, consistent and reversible fall in PAP; plasma cGMP rose from 45.5 (hypoxia) to 138 pmol/ml (hypoxia plus cigarette smoke). Neither NO nor cigarette smoke inhalation induced any change in SAP. These data suggest that exposure to cigarette smoke is able selectively to reverse acute hypoxic vasoconstriction in humans without causing systemic vasodilation, an effect likely mediated through the NO-cGMP pathway.

Topics: Administration, Inhalation; Adult; Cyclic GMP; Dose-Response Relationship, Drug; Humans; Hypoxia; Nitric Oxide; Oxygen; Pulmonary Circulation; Smoking; Vasoconstriction

To evaluate the inhibitory effect of hypoxia and atrial natriuretic peptide (ANP) on aldosterone secretion, 11 healthy male subjects were infused with 5 ng.kg-1 x min-1 ANP or placebo. The subjects were exposed in a stepwise fashion to incremental hypobaric hypoxia, which decreased arterial oxygen saturation to 79 +/- 2% in the placebo and 84 +/- 2% in the ANP condition (P < 0.05). In the placebo condition, the plasma ANP concentration increased from 13.8 +/- 1.0 to 19.6 +/- 2.3 pmol/l (P < 0.01) at the lowest barometric pressure. Plasma renin activity did not change, whereas the plasma aldosterone levels increased consequent to the increase of plasma adrenocorticotropic hormone (ACTH). Continuous infusion of ANP increased the plasma levels twofold (P < 0.001) and the level of guanosine 3',5'-cyclic monophosphate threefold (P < 0.001). However, the plasma aldosterone concentrations were not different in the two experimental conditions. Administration of supplementary oxygen significantly decreased ACTH to baseline values (P < 0.01) together with a decrease in aldosterone. Free water clearance (P = 0.05) but not sodium excretion (P = NS) increased during continuous ANP infusion. The data indicate that the aldosterone secretion in hypoxia is not inhibited by (patho)physiological plasma ANP levels. The inhibition of aldosterone secretion may well be explained by a direct effect of hypoxia on the adrenal cells. ACTH is a major stimulus of aldosterone secretion in hypoxia, which overrides the natriuretic effect of ANP.

Topics: Adrenocorticotropic Hormone; Adult; Aldosterone; Atmospheric Pressure; Atrial Natriuretic Factor; Blood Volume; Carbon Dioxide; Cyclic GMP; Humans; Hydrocortisone; Hydrogen-Ion Concentration; Hypoxia; Iodine Radioisotopes; Male; Oxygen; Renin; Single-Blind Method; Sodium

Atrial Natriuretic Peptide (ANP) is secreted in response to hypoxia and pulmonary vasoconstriction. The hormone modulates pulmonary vascular tone in vivo and decreases pulmonary edema in isolated lungs exposed to several toxic agents. In addition, ANP improves the barrier function of endothelial cell monolayers in vitro. The plasma levels of ANP are elevated in patients with high-altitude pulmonary edema. We hypothesized that under these circumstances, ANP improves pulmonary gas exchange by attenuating the transvascular permeation of plasma (water). Therefore, we studied the effect of low-dose ANP in 11 healthy mountaineers exposed to hypoxia in a single-blind, placebo-controlled, cross-over design. During four 1-h periods, the subjects were stepwise exposed to decreasing barometric pressure, with a minimum of 456 mm Hg (simulated altitude, 4,115 m). Infusion of 5 ng/kg/min human-ANP increased the plasma ANP concentrations approximately twofold. The plasma concentrations of cyclic GMP, which is the second messenger of ANP, rose approximately threefold. Infusion of ANP did not affect the hemodynamic or ventilatory response to hypoxia. The hemoglobin concentration, however, rose from 9.0 +/- 0.1 to 9.4 +/- 0.1 mmol/L (p < 0.01) during ANP infusion but not during placebo infusion. The change in plasma volume calculated from this hemoconcentration indicated that approximately 10% of the plasma volume had permeated into the interstitium. Despite the observed whole-body hemoconcentration, oxygen saturation was significantly higher during ANP infusion than during placebo infusion (84.7 +/- 1.7 versus 79.6 +/- 1.8%, p < 0.05), and the alveolar-arterial oxygen difference was significantly lower (3.5 +/- 0.7 versus 7.3 +/- 0.8 mm Hg, p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Atrial Natriuretic Factor; Blood Pressure; Blood Proteins; Carbon Dioxide; Cyclic GMP; Heart Rate; Hemoglobins; Humans; Hypoxia; Infusions, Intravenous; Mountaineering; Oxygen; Oxygen Consumption; Partial Pressure; Placebos; Plasma Volume; Pulmonary Edema; Pulmonary Gas Exchange; Single-Blind Method

Chronic hypoxia is a major driver of cardiovascular complications, including heart failure. The nitric oxide (NO) - soluble guanylyl cyclase (sGC) - cyclic guanosine monophosphate (cGMP) pathway is integral to vascular tone maintenance. Specifically, NO binds its receptor sGC within vascular smooth muscle cells (SMC) in its reduced heme (Fe

Topics: Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Guanylate Cyclase; Hypoxia; Mice; Myocytes, Smooth Muscle; Nitric Oxide; Soluble Guanylyl Cyclase

During infection, intracellular pathogens inevitably face the pressure of hypoxia. Mycobacterium tuberculosis and Mycobacterium bovis represent two typical intracellular bacteria, but the signalling pathway of their adaptation to hypoxia remains unclear. Here, we report a new mechanism of the hypoxic adaptation in M. bovis driven by the second messenger molecule c-di-GMP. We found that c-di-GMP was significantly accumulated in bacterial cells under hypoxic stress and blocked the inhibitory activity of ArgR, an arginine metabolism gene cluster regulator, which increased arginine synthesis and slowed tricarboxylic acid cycle (TCA cycle) and aerobic respiration. Meanwhile, c-di-GMP relieved the self-inhibition of argR expression, and ArgR could interact with the nitrite metabolic gene regulator Cmr, promoting the positive regulation of Cmr and, thereafter, the nitrite respiration. Consistently, c-di-GMP significantly induced the expression of arginine and nitrite metabolism gene clusters and increased the mycobacterial survival ability under hypoxia. Therefore, we found a new function of the second messenger molecule c-di-GMP and characterized ArgR as a metabolic switching regulator that can coordinate the c-di-GMP signal to trigger hypoxic adaptation in mycobacteria. Our findings provide a potential new target for blocking the life cycle of M. tuberculosis infection.

Topics: Arginine; Bacterial Proteins; Cyclic GMP; Gene Expression Regulation, Bacterial; Humans; Hypoxia; Mycobacterium bovis; Mycobacterium tuberculosis; Nitrites

The ability to detect and respond to acute oxygen (O2) shortages is indispensable to aerobic life. The molecular mechanisms and circuits underlying this capacity are poorly understood. Here, we characterize the behavioral responses of feeding Caenorhabditis elegans to approximately 1% O2. Acute hypoxia triggers a bout of turning maneuvers followed by a persistent switch to rapid forward movement as animals seek to avoid and escape hypoxia. While the behavioral responses to 1% O2 closely resemble those evoked by 21% O2, they have distinct molecular and circuit underpinnings. Disrupting phosphodiesterases (PDEs), specific G proteins, or BBSome function inhibits escape from 1% O2 due to increased cGMP signaling. A primary source of cGMP is GCY-28, the ortholog of the atrial natriuretic peptide (ANP) receptor. cGMP activates the protein kinase G EGL-4 and enhances neuroendocrine secretion to inhibit acute responses to 1% O2. Triggering a rise in cGMP optogenetically in multiple neurons, including AIA interneurons, rapidly and reversibly inhibits escape from 1% O2. Ca2+ imaging reveals that a 7% to 1% O2 stimulus evokes a Ca2+ decrease in several neurons. Defects in mitochondrial complex I (MCI) and mitochondrial complex I (MCIII), which lead to persistently high reactive oxygen species (ROS), abrogate acute hypoxia responses. In particular, repressing the expression of isp-1, which encodes the iron sulfur protein of MCIII, inhibits escape from 1% O2 without affecting responses to 21% O2. Both genetic and pharmacological up-regulation of mitochondrial ROS increase cGMP levels, which contribute to the reduced hypoxia responses. Our results implicate ROS and precise regulation of intracellular cGMP in the modulation of acute responses to hypoxia by C. elegans.

Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Calcium; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Hypoxia; Oxygen; Reactive Oxygen Species

Topics: Animals; Arginine; Cyclic GMP; Hypertension, Pulmonary; Hypoxia; Male; Myocardium; Nitric Oxide; Rats; Rats, Sprague-Dawley; Sildenafil Citrate; Vasodilator Agents

Hypoxic coronary vasospasm may lead to myocardial ischaemia and cardiac dysfunction. Inosine 3',5'-cyclic monophosphate (cIMP) is a putative second messenger to mediate this pathological process. Nevertheless, it remains unclear as to whether levels of cIMP can be regulated in living tissue such as coronary artery and if so, what is the consequence of this regulation on hypoxia-induced vasoconstriction. In the present study, we found that cIMP was a key determinant of hypoxia-induced constriction but not that of the subsequent relaxation response in porcine coronary arteries. Subsequently, coronary arteries were treated with various phosphodiesterase (PDE) inhibitors to identify PDE types that are capable of regulating cIMP levels. We found that inhibition of PDE1 and PDE5 substantially elevated cIMP content in endothelium-denuded coronary artery supplemented with exogenous purified cIMP. However, cGMP levels were far lower than their levels in intact coronary arteries and lower than cIMP levels measured in endothelium-denuded coronary arteries supplemented with exogenous cIMP. The increased cIMP levels induced by PDE1 or PDE5 inhibition further led to augmented hypoxic constriction without apparently affecting the relaxation response. In intact coronary artery, PDE1 or PDE5 inhibition up-regulated cIMP levels under hypoxic condition. Concomitantly, cGMP level increased to a comparable level. Nevertheless, the hypoxia-mediated constriction was enhanced in this situation that was largely compromised by an even stronger inhibition of PDEs. Taken together, these data suggest that cIMP levels in coronary arteries are regulated by PDE1 and PDE5, whose inhibition at a certain level leads to increased cIMP content and enhanced hypoxic constriction.

Topics: Animals; Chromatography, High Pressure Liquid; Coronary Vessels; Cyclic GMP; Cyclic IMP; Cyclic Nucleotide Phosphodiesterases, Type 1; Cyclic Nucleotide Phosphodiesterases, Type 5; Endothelium, Vascular; Hypoxia; Metabolomics; Nitric Oxide; Phosphodiesterase 5 Inhibitors; Swine; Tandem Mass Spectrometry; Vasoconstriction

Nitric oxide (NO) donors may be useful for treating pulmonary hypertension (PH) complicating sickle cell disease (SCD), as endogenous NO is inactivated by hemoglobin released by intravascular hemolysis. Here, we investigated the effects of the new NO donor NCX1443 on PH in transgenic SAD mice, which exhibit mild SCD without severe hemolytic anemia. In SAD and wild-type (WT) mice, the pulmonary pressure response to acute hypoxia was similar and was abolished by 100 mg/kg NCX1443. The level of PH was also similar in SAD and WT mice exposed to chronic hypoxia (9% O2) alone or with SU5416 and was similarly reduced by daily NCX1443 gavage. Compared with WT mice, SAD mice exhibited higher levels of HO-1, endothelial NO synthase, and PDE5 but similar levels of lung cyclic guanosine monophosphate. Cultured pulmonary artery smooth muscle cells from SAD mice grew faster than those from WT mice and had higher PDE5 protein levels. Combining NCX1443 and a PDE5 inhibitor suppressed the growth rate difference between SAD and WT cells and induced a larger reduction in hypoxic PH severity in SAD than in WT mice. By amplifying endogenous protective mechanisms, NCX1443 in combination with PDE5 inhibition may prove useful for treating PH complicating SCD.

Topics: Anemia, Sickle Cell; Animals; Antihypertensive Agents; Arterial Pressure; Cell Proliferation; Cells, Cultured; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Disease Models, Animal; Heme Oxygenase-1; Hypertension, Pulmonary; Hypoxia; Male; Membrane Proteins; Mice, Inbred C57BL; Mice, Transgenic; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type III; Phosphodiesterase 5 Inhibitors; Pulmonary Artery

Adequate perfusion of the placental vasculature is essential to meet the metabolic demands of fetal growth and development. Lacking neural control, local tissue metabolites, circulating and physical factors contribute significantly to blood flow regulation. Nitric oxide (NO) is a key regulator of fetoplacental vascular tone. Nitrite, previously considered an inert end-product of NO oxidation, has been shown to provide an important source of NO. Reduction of nitrite to NO may be particularly relevant in tissue when the oxygen-dependent NO synthase (NOS) activity is compromised, e.g. in hypoxia. The contribution of this pathway in the placenta is currently unknown. We hypothesised that nitrite vasodilates human placental blood vessels, with enhanced efficacy under hypoxia. Placentas were collected from uncomplicated pregnancies and the vasorelaxant effect of nitrite (10

Topics: Adult; Arteries; Benzoates; Chorion; Cyclic GMP; Dose-Response Relationship, Drug; Female; Humans; Hypoxia; Imidazoles; Nitrites; Placenta; Pregnancy; Sodium Nitrite; Vasodilation; Veins

Persistent pulmonary hypertension of the newborn (PPHN) features hypoxemia, pulmonary vasoconstriction, and impaired cardiac inotropy. We previously reported low basal and stimulated cAMP in hypoxic pulmonary artery smooth muscle cells (PASMCs). We now examine pulmonary arterial adenylyl cyclase (AC) activity and regulation in hypoxic PPHN. PPHN was induced in newborn swine by normobaric hypoxia (fraction of inspired oxygen 0.10) for 72 h and compared with age-matched normoxic controls. We studied relaxation of pulmonary arterial (PA) rings to AC activator forskolin and cGMP activator sodium nitroprusside (SNP) by isometric myography, ATP content, phosphodiesterase activity, AC content, isoform expression, and catalytic activity in presence or absence of Gαs-coupled receptor agonists, forskolin, or transnitrosylating agents in human and neonatal porcine PASMCs and HEK293T stably expressing AC isoform 6, after 72 h hypoxia (10% O

Topics: Adenylyl Cyclases; Animals; Animals, Newborn; Cell Line; Cyclic GMP; Down-Regulation; HEK293 Cells; Humans; Hypertension, Pulmonary; Hypoxia; Muscle Relaxation; Myocytes, Smooth Muscle; Nitroprusside; Persistent Fetal Circulation Syndrome; Pulmonary Artery; Swine

Neonatal pulmonary vascular disease (PVD) is increasingly recognized as a disease that complicates the cardiopulmonary adaptations after birth and predisposes to long-term cardiopulmonary disease. There is growing evidence that PVD is associated with disruptions in the nitric oxide (NO)-cGMP-phosphodiesterase 5 (PDE5) pathway. Examination of the functionality of different parts of this pathway is required for better understanding of the pathogenesis of neonatal PVD. For this purpose, the role of the NO-cGMP-PDE5 pathway in regulation of pulmonary vascular function was investigated in vivo, both at rest and during exercise, and in isolated pulmonary small arteries in vitro, in a neonatal swine model with hypoxia-induced PVD. Endothelium-dependent vasodilatation was impaired in piglets with hypoxia-induced PVD both in vivo at rest and in vitro. Moreover, the responsiveness to the NO-donor SNP was reduced in hypoxia-exposed piglets in vivo, while the relaxation to SNP and 8-bromo-cyclicGMP in vitro were unaltered. Finally, PDE5 inhibition-induced pulmonary vasodilatation was impaired in hypoxia-exposed piglets both in vitro and in vivo at rest. During exercise, however, the pulmonary vasodilator effect of PDE5 inhibition was significantly larger in hypoxia-exposed as compared to normoxia-exposed piglets. In conclusion, the impaired endothelium-dependent vasodilatation in piglets with hypoxia-induced PVD was accompanied by reduced responsiveness to NO, potentially caused by altered sensitivity and/or activity of soluble guanylyl cyclase (sGC), resulting in an impaired cGMP production. Our findings in a newborn animal model for neonatal PVD suggests that sGC stimulators/activators may be a novel treatment strategy to alleviate neonatal PVD.

Topics: Animals; Bronchopulmonary Dysplasia; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Endothelium, Vascular; Female; Guanylate Cyclase; Hypoxia; Lung; Male; Nitric Oxide; Phosphodiesterase 5 Inhibitors; Physical Conditioning, Animal; Swine; Vasodilation

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Colonic Neoplasms; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Drug Resistance, Neoplasm; Drug Synergism; Drugs, Chinese Herbal; Humans; Hypoxia; Male; Mice, Inbred BALB C; Nitroso Compounds; Oxadiazoles; Oxazines; Phosphodiesterase 5 Inhibitors; Plant Extracts; Reactive Oxygen Species; Serum Albumin, Human; Soluble Guanylyl Cyclase; Vardenafil Dihydrochloride

Elevation of hemoglobin concentration, a common adaptive response to high-altitude hypoxia, occurs among Oromo but is dampened among Amhara highlanders of East Africa. We hypothesized that Amhara highlanders offset their smaller hemoglobin response with a vascular response. We tested this by comparing Amhara and Oromo highlanders at 3,700 and 4,000 m to their lowland counterparts at 1,200 and 1,700 m. To evaluate vascular responses, we assessed urinary levels of nitrate (NO

Topics: Adaptation, Physiological; Africa, Eastern; Altitude; Altitude Sickness; Blood Pressure; Blood Vessels; Cyclic GMP; Demography; Diastole; Ethnicity; Hemoglobins; Humans; Hypoxia; Nitrates; Oxyhemoglobins

Retinal hypoxia and oxidative stress are involved in several retinal degenerations including diabetic retinopathy, glaucoma, central retinal artery occlusion, or retinopathy of prematurity. The second messenger cyclic guanosine monophosphate (cGMP) has been reported to be protective for neuronal cells under several pathological conditions including ischemia/hypoxia. The purpose of this study was to evaluate whether the accumulation of cGMP through the pharmacological inhibition of phosphodiesterase (PDE) with Zaprinast prevented retinal degeneration induced by mild hypoxia in cultures of porcine retina. Exposure to mild hypoxia (5% O2) for 24h reduced cGMP content and induced retinal degeneration by caspase dependent and independent (PARP activation) mechanisms. Hypoxia also produced a redox imbalance reducing antioxidant response (superoxide dismutase and catalase activities) and increasing superoxide free radical release. Zaprinast reduced mild hypoxia-induced cell death through inhibition of caspase-3 or PARP activation depending on the cell layer. PDE inhibition also ameliorated the effects of mild hypoxia on antioxidant response and the release of superoxide radical in the photoreceptor layer. The use of a PKG inhibitor, KT5823, suggested that cGMP-PKG pathway is involved in cell survival and antioxidant response. The inhibition of PDE, therefore, could be useful for reducing retinal degeneration under hypoxic/ischemic conditions.

Topics: Animals; Antioxidants; Caspase 3; Cell Death; Cyclic GMP; Gene Expression Regulation; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Lactic Acid; Oxidative Stress; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Poly(ADP-ribose) Polymerases; Pyruvic Acid; Retina; Superoxides; Swine; Tissue Culture Techniques

Nitric oxide (NO) acts as essential regulator of vasculogenesis and angiogenesis and is critical for arteriogenesis. Whether NO's effects in vivo are mediated through NO-sensitive guanylyl cyclase (NO-GC) and thus by cGMP-dependent mechanisms has been only poorly addressed. Mice lacking NO-GC globally or specifically in smooth muscle cells (SMC) or endothelial cells (EC) were subjected to two established models for arteriogenesis and angiogenesis, namely hindlimb ischemia and oxygen-induced retinopathy. Our data clearly show the involvement of NO-GC in the recovery of blood flow after hindlimb ischemia, and this effect could be attributed to NO-GC in SMC. In the retina, global deletion of NO-GC led to reduced oxygen-induced vessel loss and hypoxia-induced capillary regrowth, whereas pathological neovascularization was increased. These effects were also seen in mice with SMC-specific NO-GC deletion but not in animals lacking NO-GC in EC. Intriguingly, NO-GC was found to be strongly expressed in retinal pericytes. Our data prove the involvement of NO-GC in growth and plasticity of hindlimb and retinal vasculature after ischemic/hypoxic insult.

Topics: Animals; Cyclic GMP; Endothelial Cells; Exons; Guanylate Cyclase; Hindlimb; Hypoxia; Image Processing, Computer-Assisted; Immunohistochemistry; Ischemia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocytes, Smooth Muscle; Neovascularization, Pathologic; Nitric Oxide; Oxygen; Pericytes; Receptors, Cytoplasmic and Nuclear; Retina; Retinal Diseases; Signal Transduction; Soluble Guanylyl Cyclase; Time Factors

It has been suggested that increase in acute nitric oxide (NO) or cyclic guanosine monophosphate production may be involved in cardioprotection induced by chronic hypoxia (CH). We studied the effect of NO donor molsidomine and phosphodiesterase type 5 inhibitor sildenafil on myocardial ischemia/reperfusion (I/R) injury in rats adapted to CH. Male Wistar rats were exposed to continuous hypoxia in a normobaric chamber (10 % O(2), 4 weeks). Rats received either saline, molsidomine (10 mg/kg body weight, i.v.) or sildenafil (0.7 mg/kg body weight, i.v.) 30 min before ischemia. Control rats were kept under normoxia and treated in a corresponding manner. Adaptation to CH increased the myocardial ischemic tolerance. Acute treatment with either molsidomine or sildenafil significantly reduced infarct size in normoxic rats and further enhanced cardioprotection induced by CH. However, the cardioprotective effect of CH on I/R injury was not additive to the cardioprotection provided by the drugs.

Topics: Animals; Chronic Disease; Cyclic GMP; Hypoxia; Male; Myocardial Ischemia; Nitric Oxide; Rats; Rats, Wistar; Signal Transduction

Inhaled nitric oxide (NO) and other cGMP- or cAMP-dependent pulmonary vasodilators are often used in combination for the treatment of the persistent pulmonary hypertension of the newborn syndrome. There is in vitro evidence to indicate that NO downregulate the pulmonary vascular response to cGMP-dependent agonists raising concern as to whether a synergistic effect is observed when employing a combined strategy in newborns. Hypothesizing that a synergistic effect is absent, we evaluated newborn and juvenile rat pulmonary arteries to determine the individual and combined vasodilatory effect of cGMP- and cAMP-dependent agonists. In precontracted near-resistance pulmonary arteries, the addition of sildenafil reduced vasorelaxation response to NO donor S-nitroso-N-acetyl penicillamine (SNAP). A similar decrease in SNAP-induced vasodilation was observed in arteries pretreated with BAY 41-2272 (10(-9) M), a soluble guanylate cyclase stimulator cGMP, and its downstream protein kinase activator. cGMP also reduced the vasorelaxant response to the cAMP-dependent forskolin. Inhibition of endogenous vascular NO generation enhanced SNAP-induced relaxation. The present data suggest that the mechanism involved in the cGMP desensitization to other relaxant agonists involves downregulation of the small heat shock protein HSP20 and is evident in rat pulmonary and systemic vascular smooth muscle cells. In newborn rats with chronic hypoxia-induced pulmonary hypertension, the combination of sildenafil and inhaled NO resulted in a lesser reduction in pulmonary vascular resistance compared with their individual effect. These data suggest that clinical exposure to one cGMP-dependent pulmonary vasodilator may affect the response to other cGMP- or cAMP-mediated agonists.

Topics: Animals; Animals, Newborn; Chronic Disease; Cyclic GMP; Female; HSP20 Heat-Shock Proteins; HSP27 Heat-Shock Proteins; Hypoxia; Male; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Donors; Phosphorylation; Piperazines; Primary Cell Culture; Protein Kinase Inhibitors; Pulmonary Circulation; Purines; Rats; Rats, Sprague-Dawley; Sildenafil Citrate; Sulfones; Vasodilation; Vasodilator Agents

Insulin and inflammatory cytokines may be involved in equine laminitis, which might be associated with digital vascular dysfunction. This study determined the effects of TNF-α and insulin on the endothelial-dependent relaxant responses of equine digital blood vessels and on equine digital vein endothelial cell (EDVEC) cGMP production.. Isolated rings of equine digital arteries (EDAs) and veins (EDVs) were obtained and EDVECs were cultured from horses euthanized at an abattoir.. The effect of incubation with TNF-α (10 ng/ml) and/or insulin (1,000 μIU/ml) for 1.5 h or overnight under hyperoxic and hypoxic conditions on carbachol (endothelium-dependent) induced relaxation was assessed. The time course and concentration dependency of the effect of TNF-α and the effect of insulin (1,000 μIU/ml) on EDVEC cGMP production was determined.. Incubation of EDAs overnight with TNF-α under hypoxic conditions resulted in endothelial-dependent vascular dysfunction. EDVs produced a more variable response. TNF-α increased EDVEC cGMP formation in a time- and concentration-dependent manner. Insulin had no significant effects.. There is a mismatch between the results obtained from isolated vessel rings and cultured endothelial cells suggesting TNF-α may reduce the biological effect of NO by reducing its bioavailability rather than its formation, leading to endothelial cell dysregulation.

Topics: Animals; Arteries; Carbachol; Cells, Cultured; Cyclic GMP; Endothelial Cells; Horses; Hyperoxia; Hypoxia; In Vitro Techniques; Insulin; Metatarsophalangeal Joint; Tumor Necrosis Factor-alpha; Vasodilation; Veins

Hypoxia or hypoxia/reoxygenation impairs nitric oxide (NO)-mediated relaxation through the increase in superoxide generation in monkey coronary arteries. Soluble guanylate cyclase (sGC), the target enzyme of NO, has been shown to change from the NO-sensitive reduced form to the NO-insensitive oxidized/heme-free form under substantial oxidative stress, so the present study investigated whether hypoxia or hypoxia/reoxygenation influences sGC redox equilibrium. In isolated monkey coronary arteries without endothelium, the relaxation caused by the sGC stimulator BAY 41-2272 (Emax: 93.3% ± 2.2%) was somewhat impaired under hypoxia (Emax: 86.3% ± 2.6%) or hypoxia/reoxygenation (Emax: 86.1% ± 3.2%), whereas that by the sGC activator BAY 60-2770 (Emax: 86.0% ± 3.2%) was significantly augmented under hypoxia (Emax: 94.4% ± 1.3%) or hypoxia/reoxygenation (Emax: 95.5% ± 1.1%). In addition, cGMP formation in response to BAY 41-2272 and BAY 60-2770 was inhibited and stimulated, respectively, under hypoxia or hypoxia/reoxygenation. The effects of hypoxia or hypoxia/reoxygenation on BAY 41-2272- and BAY 60-2770-induced vasorelaxation were completely canceled by the treatment with the superoxide dismutase mimetic tempol. These findings suggest that sGC redox equilibrium in the coronary artery is shifted towards the NO-insensitive form under hypoxia or hypoxia/reoxygenation and that superoxide seems to play an important role in this shift.

Topics: Animals; Benzoates; Biphenyl Compounds; Coronary Vessels; Cyclic GMP; Female; Guanylate Cyclase; Hydrocarbons, Fluorinated; Hypoxia; In Vitro Techniques; Macaca; Male; Nitric Oxide; Oxidation-Reduction; Oxidative Stress; Pyrazoles; Pyridines; Solubility; Superoxide Dismutase; Superoxides; Vasodilation

The NOS (nitric oxide synthase) inhibitor ADMA (asymmetric dimethylarginine) contributes to the pathogenesis of pulmonary hypertension. Reduced levels of the enzymes metabolizing ADMA, dimethylarginine dimethylaminohydrolases (DDAH1 and DDAH2) and increased levels of miR-21 are linked to disease pathology, but the mechanisms are not understood. In the present study we assessed the potential role of miR-21 in the regulation of hypoxia-induced changes in ADMA metabolism in vitro and in vivo. Hypoxia inhibited DDAH1 and DDAH2 expression and increased ADMA levels in cultured human pulmonary endothelial cells. In contrast, in human pulmonary smooth muscle cells, only DDAH2 was reduced whereas ADMA levels remained unchanged. Endothelium-specific down-regulation of DDAH1 by miR-21 in hypoxia induced endothelial dysfunction and was prevented by overexpression of DDAH1 and miR-21 blockade. DDAH1, but not DDAH2, mRNA levels were reduced, whereas miR-21 levels were elevated in lung tissues from patients with pulmonary arterial hypertension and mice with pulmonary hypertension exposed to 2 weeks of hypoxia. Hypoxic mice treated with miR-21 inhibitors and DDAH1 transgenic mice showed elevated lung DDAH1, increased cGMP levels and attenuated pulmonary hypertension. Regulation of DDAH1 by miR-21 plays a role in the development of hypoxia-induced pulmonary hypertension and may be of broader significance in pulmonary hypertension.

Topics: Amidohydrolases; Animals; Arginine; Cells, Cultured; Cyclic GMP; Endothelial Cells; Familial Primary Pulmonary Hypertension; Humans; Hypertension, Pulmonary; Hypoxia; Lung; Male; Mice; Mice, Transgenic; MicroRNAs; Myocytes, Smooth Muscle

Exposing mice to a chronic hypoxic treatment (10% oxygen, 21 days) that promotes pulmonary hypertension was observed to attenuate the pulmonary vasoconstriction response to acute hypoxia (HPV) both in vivo and in isolated pulmonary arteries. Since catalase restored the HPV response in isolated arteries, it appeared to be attenuated by extracellular hydrogen peroxide. Chronic hypoxia promoted the detection of elevated lung superoxide, extracellular peroxide, extracellular SOD expression, and protein kinase G (PKG) activation [based on PKG dimerization and vasodilator-stimulated phosphoprotein (VASP) phosphorylation], suggesting increased generation of extracellular peroxide and PKG activation may contribute to the suppression of HPV. Aorta from mice exposed to 21 days of hypoxia also showed evidence for extracellular hydrogen peroxide, suppressing the relaxation response to acute hypoxia. Peroxide appeared to partially suppress contractions to phenylephrine used in the study of in vitro hypoxic responses. Treatment of mice with the heme precursor δ-aminolevulinic acid (ALA; 50 mg·kg(-1)·day(-1)) during exposure to chronic hypoxia was examined as a pulmonary hypertension therapy because it could potentially activate beneficial cGMP-mediated effects through promoting a prolonged protoporphyrin IX (PpIX)-elicited activation of soluble guanylate cyclase. ALA attenuated pulmonary hypertension, increases in both superoxide and peroxide, and the suppression of in vitro and in vivo HPV responses. ALA generated prolonged detectible increases in PpIX and PKG-associated phosphorylation of VASP, suggesting PKG activation may contribute to suppression of pulmonary hypertension and prevention of alterations in extracellular peroxide that appear to be attenuating HPV responses caused by chronic hypoxia.

Topics: Acute Disease; Aminolevulinic Acid; Animals; Antihypertensive Agents; Aorta; Cell Adhesion Molecules; Chronic Disease; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Enzyme Activation; Familial Primary Pulmonary Hypertension; Hydrogen Peroxide; Hypertension, Pulmonary; Hypoxia; Male; Mice; Mice, Inbred C57BL; Microfilament Proteins; Phosphoproteins; Phosphorylation; Protoporphyrins; Pulmonary Artery; Superoxide Dismutase; Superoxides; Time Factors; Up-Regulation; Vasoconstriction

Adverse events in utero are associated with the occurrence of chronic diseases in adulthood. We previously demonstrated in mice that perinatal hypoxia resulted in altered pulmonary circulation in adulthood, with a decreased endothelium-dependent relaxation of pulmonary arteries, associated with long-term alterations in the nitric oxide (NO)/cyclic GMP pathway. The present study investigated whether inhaled NO (iNO) administered simultaneously to perinatal hypoxia could have potential beneficial effects on the adult pulmonary circulation. Indeed, iNO is the therapy of choice in humans presenting neonatal pulmonary hypertension. Long-term effects of neonatal iNO therapy on adult pulmonary circulation have not yet been investigated. Pregnant mice were placed in hypoxia (13% O2) with simultaneous administration of iNO 5 days before delivery until 5 days after birth. Pups were then raised in normoxia until adulthood. Perinatal iNO administration completely restored acetylcholine-induced relaxation, as well as endothelial nitric oxide synthase protein content, in isolated pulmonary arteries of adult mice born in hypoxia. Right ventricular hypertrophy observed in old mice born in hypoxia compared to controls was also prevented by perinatal iNO treatment. Therefore, simultaneous administration of iNO during perinatal hypoxic exposure seems able to prevent adverse effects of perinatal hypoxia on the adult pulmonary circulation.

Topics: Administration, Inhalation; Adult; Animals; Cyclic GMP; Female; Humans; Hypertrophy, Right Ventricular; Hypoxia; Mice; Nitric Oxide; Oxygen; Pregnancy; Pulmonary Circulation; Signal Transduction

Excess superoxide has been implicated in pulmonary hypertension (PH). We previously found lung overexpression of the antioxidant extracellular superoxide dismutase (EC-SOD) attenuates PH and pulmonary artery (PA) remodeling. Although comprising a small fraction of total SOD activity in most tissues, EC-SOD is abundant in arteries. We hypothesize that the selective loss of vascular EC-SOD promotes hypoxia-induced PH through redox-sensitive signaling pathways. EC-SOD(loxp/loxp) × Tg(cre/SMMHC) mice (SMC EC-SOD KO) received tamoxifen to conditionally deplete smooth muscle cell (SMC)-derived EC-SOD. Mice were exposed to hypobaric hypoxia for 35 days, and PH was assessed by right ventricular systolic pressure measurements and right ventricle hypertrophy. Vascular remodeling was evaluated by morphometric analysis and two-photon microscopy for collagen. We examined cGMP content and soluble guanylate cyclase expression and activity in lung, lung phosphodiesterase 5 (PDE5) expression and activity, and expression of endothelial nitric oxide synthase and GTP cyclohydrolase-1 (GTPCH-1), the rate-limiting enzyme in tetrahydrobiopterin synthesis. Knockout of SMC EC-SOD selectively decreased PA EC-SOD without altering total lung EC-SOD. PH and vascular remodeling induced by chronic hypoxia was augmented in SMC EC-SOD KO. Depletion of SMC EC-SOD did not impact content or activity of lung soluble guanylate cyclase or PDE5, yet it blunted the hypoxia-induced increase in cGMP. Although total eNOS was not altered, active eNOS and GTPCH-1 decreased with hypoxia only in SMC EC-SOD KO. We conclude that the localized loss of PA EC-SOD augments chronic hypoxic PH. In addition to oxidative inactivation of NO, deletion of EC-SOD seems to reduce eNOS activity, further compromising pulmonary vascular function.

Topics: Animals; Blood Pressure; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Estrogen Antagonists; GTP Cyclohydrolase; Guanylate Cyclase; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Lung; Mice; Mice, Knockout; Nitric Oxide Synthase Type III; Pulmonary Artery; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Soluble Guanylyl Cyclase; Superoxide Dismutase; Tamoxifen

Chronic hypoxia attenuates soluble guanylate cyclase-induced vasorelaxation in serotonin (5-HT)-contracted ovine carotid arteries. Because protein kinase G (PKG) mediates many effects of soluble guanylate cyclase activation through phosphorylation of multiple kinase targets in vascular smooth muscle, we tested the hypothesis that chronic hypoxia reduces the ability of PKG to phosphorylate its target proteins, which attenuates the ability of PKG to induce vasorelaxation. We also tested the hypothesis that hypoxia attenuates PKG expression and/or activity. Arteries from normoxic and chronically hypoxic (altitude of 3,820 m for 110 days) fetal and adult sheep were denuded of endothelium and equilibrated with 95% O2-5% CO2 in the presence of nitro-l-arginine methyl ester (l-NAME) and N(G)-nitro-l-arginine (l-NNA) to inhibit residual endothelial nitric oxide synthase. Concentration-response relations for 5-HT were determined in the presence of prazosin to minimize activation of α-adrenergic receptors. The PKG activator 8-(p-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCTP-cGMP) reduced agonist binding affinity of the 5-HT receptor in a concentration-dependent manner that was attenuated by hypoxia. Expression and activity of PKG-I was not significantly affected by chronic hypoxia in either fetal or adult arteries, although PKG-I abundance was greater in fetal arteries. Pretreatment with the large conductance calcium-sensitive potassium channel (BK) inhibitor iberiotoxin attenuated the vasorelaxation induced by 8-pCPT-cGMP in normoxic but not chronically hypoxic arteries. These results support the hypothesis that hypoxia attenuates the vasorelaxant effects of PKG through suppression of the ability of PKG to activate large conductance calcium-sensitive potassium channels in arterial smooth muscle. The results also reveal that this hypoxic effect is greater in fetal than adult arteries and that chronic maternal hypoxia can profoundly affect fetal vascular function.

Topics: Aging; Animals; Blotting, Western; Carotid Arteries; Chronic Disease; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Endpoint Determination; Female; Fetus; Hypoxia; Large-Conductance Calcium-Activated Potassium Channels; Muscle, Smooth, Vascular; Phosphorylation; Pregnancy; Receptor, Serotonin, 5-HT2A; Serotonin; Serotonin Receptor Agonists; Sheep; Thionucleotides

Transient receptor potential canonical (TRPC) proteins play important roles in chronically hypoxic pulmonary hypertension (CHPH). Previous results indicated that sildenafil inhibited TRPC1 and TRPC6 expression in rat distal pulmonary arteries (PAs). However, the underlying mechanisms remain unknown. We undertook this study to investigate the downstream signaling of sildenafil's regulation on TRPC1 and TRPC6 expression in pulmonary arterial smooth muscle cells (PASMCs). Hypoxia-exposed rats (10% O2 for 21 d) and rat distal PASMCs (4% O2 for 60 h) were taken as models to mimic CHPH. Real-time PCR, Western blotting, and Fura-2-based fluorescent microscopy were performed for mRNA, protein, and Ca(2+) measurements, respectively. The cellular cyclic guanosine monophosphate (cGMP) analogue 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate sodium salt (CPT-cGMP) (100 μM) inhibited TRPC1 and TRPC6 expression, store-operated Ca(2+) entry (SOCE), and the proliferation and migration of PASMCs exposed to prolonged hypoxia. The inhibition of CPT-cGMP on TRPC1 and TRPC6 expression in PASMCs was relieved by either the inhibition or knockdown of cGMP-dependent protein kinase (PKG) and peroxisome proliferator-activated receptor γ (PPARγ) expression. Under hypoxic conditions, CPT-cGMP increased PPARγ expression. This increase was abolished by the PKG antagonists Rp8 or KT5823. PPARγ agonist GW1929 significantly decreased TRPC1 and TRPC6 expression in PASMCs. Moreover, hypoxia exposure decreased, whereas sildenafil treatment increased, PKG and PPARγ expression in PASMCs ex vivo, and in rat distal PAs in vivo. The suppressive effects of sildenafil on TRPC1 and TRPC6 in rat distal PAs and on the hemodynamic parameters of CHPH were inhibited by treatment with the PPARγ antagonist T0070907. We conclude that sildenafil inhibits TRPC1 and TRPC6 expression in PASMCs via cGMP-PKG-PPARγ-dependent signaling during CHPH.

Topics: Animals; Benzamides; Benzophenones; Carbazoles; Cell Movement; Cell Proliferation; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Gene Expression Regulation; Hypoxia; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphodiesterase 5 Inhibitors; Piperazines; PPAR gamma; Protein Kinase Inhibitors; Pulmonary Artery; Purines; Pyridines; Rats; Rats, Wistar; Signal Transduction; Sildenafil Citrate; Sulfones; TRPC Cation Channels; Tyrosine

Acute and sustained hypoxic pulmonary vasoconstriction (HPV), as well as chronic pulmonary hypertension (PH), is modulated by nitric oxide (NO). NO synthesis can be decreased by asymmetric dimethylarginine (ADMA), which is degraded by dimethylarginine dimethylaminohydrolase-1 (DDAH1). We investigated the effects of DDAH1 overexpression (DDAH1(tg)) on HPV and chronic hypoxia-induced PH. HPV was measured during acute (10 min) and sustained (3 h) hypoxia in isolated mouse lungs. Chronic PH was induced by the exposure of mice to 4 weeks of hypoxia. ADMA and cyclic 3',5'-guanosine monophosphate (cGMP) were determined by ELISA, and NO generation was determined by chemiluminescence. DDAH1 overexpression exerted no effects on acute HPV. However, DDAH1(tg) mice showed decreased sustained HPV compared with wild-type (WT) mice. Concomitantly, ADMA was decreased, and concentrations of NO and cGMP were significantly increased in DDAH1(tg). The administration of either Nω-nitro-l-arginine or 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one potentiated sustained HPV and partly abolished the differences in sustained HPV between WT and DDAH1(tg) mice. The overexpression of DDAH1 exerted no effect on the development of chronic hypoxia-induced PH. DDAH1 overexpression selectively decreased the sustained phase of HPV, partly via activation of the NO-cGMP pathway. Thus, increased ADMA concentrations modulate sustained HPV, but not acute HPV or chronic hypoxia-induced PH.

Topics: Amidohydrolases; Animals; Arginine; Blood Vessels; Cyclic GMP; Gene Expression; Hemodynamics; Hypertension, Pulmonary; Hypoxia; Lung; Mice; Nitric Oxide; Nitroarginine; Organ Culture Techniques; Oxadiazoles; Signal Transduction; Vasoconstriction

Hypoxia causes a regulated decrease in body temperature (Tb), a response that has been aptly called anapyrexia, but the mechanisms involved are not completely understood. The roles played by nitric oxide (NO) and other neurotransmitters have been documented during hypoxia-induced anapyrexia, but no information exists with respect to hydrogen sulfide (H(2)S), a gaseous molecule endogenously produced by cystathionine β-synthase (CBS). We tested the hypothesis that H(2)S production is enhanced during hypoxia and that the gas acts in the anteroventral preoptic region (AVPO; the most important thermosensitive and thermointegrative region of the CNS) modulating hypoxia-induced anapyrexia. Thus, we assessed CBS and nitric oxide synthase (NOS) activities [by means of H(2)S and nitrite/nitrate (NO(x)) production, respectively] as well as cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) levels in the anteroventral third ventricle region (AV3V; where the AVPO is located) during normoxia and hypoxia. Furthermore, we evaluated the effects of pharmacological modifiers of the H(2)S pathway given i.c.v. or intra-AVPO. I.c.v. or intra-AVPO microinjection of CBS inhibitor caused no change in Tb under normoxia but significantly attenuated hypoxia-induced anapyrexia. During hypoxia there were concurrent increases in H(2)S production, which could be prevented by CBS inhibitor, indicating the endogenous source of the gas. cAMP concentration, but not cGMP and NO(x), correlated with CBS activity. CBS inhibition increased NOS activity, whereas H(2)S donor decreased NO(x) production. In conclusion, hypoxia activates H(2)S endogenous production through the CBS-H(2)S pathway in the AVPO, having a cryogenic effect. Moreover, the present data are consistent with the notion that the two gaseous molecules, H(2)S and NO, play a key role in mediating the drop in Tb caused by hypoxia and that a fine-balanced interplay between NOS-NO and CBS-H(2)S pathways takes place in the AVPO of rats exposed to hypoxia.

Topics: Aminooxyacetic Acid; Analysis of Variance; Animals; Body Temperature; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hydrogen Sulfide; Hypothermia; Hypoxia; Male; Microinjections; Nitrates; Nitric Oxide Synthase; Nitrites; Preoptic Area; Rats; Rats, Wistar; Sulfides; Third Ventricle; Time Factors

The nitrite anion represents the circulatory and tissue storage form of nitric oxide (NO) and a signaling molecule, capable of conferring cardioprotection and many other health benefits. However, molecular mechanisms for observed cardioprotective properties of nitrite remain largely unknown. We have evaluated the NO-like bioactivity and cardioprotective efficacies of sodium nitrite supplemented in drinking water in rats exposed to short-term chronic hypobaric hypoxia. We observed that, nitrite significantly attenuates hypoxia-induced oxidative stress, modulates HIF-1α stability and promotes NO-cGMP signaling in hypoxic heart. To elucidate potential downstream targets of nitrite during hypoxia, we performed a microarray analysis of nitrite supplemented hypoxic hearts and compared with both hypoxic and nitrite supplemented normoxic hearts respectively. The analysis revealed a significant increase in the expression of many antioxidant genes, transcription factors and cardioprotective signaling pathways which was subsequently confirmed by qRT-PCR and Western blotting. Conversely, hypoxia exposure increased oxidative stress, activated inflammatory cytokines, downregulated ion channels and altered expression of both pro- and anti-oxidant genes. Our results illustrate the physiological function of nitrite as an eNOS-independent source of NO in heart profoundly modulating the oxidative status and cardiac transcriptome during hypoxia.

Topics: Animals; Antioxidants; Cyclic GMP; Dietary Supplements; Gene Expression Regulation; Heart; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Myocardium; Nitric Oxide; Nitric Oxide Synthase Type III; Nitrites; Oxidative Stress; Rats; Rats, Sprague-Dawley; Signal Transduction; Transcriptome

Nitric oxide (NO) is an important vascular modulator in the development of pulmonary hypertension. NO exerts its regulatory effect mainly by activating soluble guanylate cyclase (sGC) to synthesize cyclic guanosine monophosphate (cGMP). Exposure to hypoxia causes pulmonary hypertension. But in lung disease, hypoxia is commonly accompanied by hypercapnia. The aim of this study was to examine the changes of sGC enzyme activity and cGMP content in lung tissue, as well as the expression of inducible nitric oxide synthase (iNOS) and sGC in rat pulmonary artery after exposure to hypoxia and hypercapnia, and assess the role of iNOS-sGC-cGMP signal pathway in the development of hypoxic and hypercapnic pulmonary hypertension. Male Sprague-Dawley rats were exposed to hypoxia and hypercapnia for 4 weeks to establish model of chronic pulmonary hypertension. Weight-matched rats exposed to normoxia served as control. After exposure to hypoxia and hypercapnia, mean pulmonary artery pressure, the ratio of right ventricle/left ventricle+septum, and the ratio of right ventricle/body weight were significantly increased. iNOS mRNA and protein levels were significantly increased, but sGC α(1) mRNA and protein levels were significantly decreased in small pulmonary arteries of hypoxic and hypercapnic exposed rat. In addition, basal and stimulated sGC enzyme activity and cGMP content in lung tissue were significantly lower after exposure to hypoxia and hypercapnia. These results demonstrate that hypoxia and hypercapnia lead to the upregulation of iNOS expression, downregulation of sGC expression and activity, which then contribute to the development of pulmonary hypertension.

Topics: Animals; Cyclic GMP; Guanylate Cyclase; Heart Ventricles; Hypercapnia; Hypertension, Pulmonary; Hypoxia; Lung; Male; Nitric Oxide; Nitric Oxide Synthase Type II; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Soluble Guanylyl Cyclase; Up-Regulation; Ventricular Function

Pulmonary hypertension (PH) is a multifactorial disease characterized by increased pulmonary vascular resistance and right ventricular failure; morbidity and mortality remain unacceptably high. Loss of nitric oxide (NO) bioactivity is thought to contribute to the pathogenesis of PH, and agents that augment pulmonary NO signaling are clinically effective in the disease. Inorganic nitrate (NO(3)(-)) and nitrite (NO(2)(-)) elicit a reduction in systemic blood pressure in healthy individuals; this effect is underpinned by endogenous and sequential reduction to NO. Herein, we determined whether dietary nitrate and nitrite might be preferentially reduced to NO by the hypoxia associated with PH, and thereby offer a convenient, inexpensive method of supplementing NO functionality to reduce disease severity.. Dietary nitrate reduced the right ventricular pressure and hypertrophy, and pulmonary vascular remodeling in wild-type mice exposed to 3 weeks of hypoxia; this beneficial activity was mirrored largely by dietary nitrite. The cytoprotective effects of dietary nitrate were associated with increased plasma and lung concentrations of nitrite and cGMP. The beneficial effects of dietary nitrate and nitrite were reduced in mice lacking endothelial NO synthase or treated with the xanthine oxidoreductase inhibitor allopurinol.. These data demonstrate that dietary nitrate, and to a lesser extent dietary nitrite, elicit pulmonary dilatation, prevent pulmonary vascular remodeling, and reduce the right ventricular hypertrophy characteristic of PH. This favorable pharmacodynamic profile depends on endothelial NO synthase and xanthine oxidoreductase -catalyzed reduction of nitrite to NO. Exploitation of this mechanism (ie, dietary nitrate/nitrite supplementation) represents a viable, orally active therapy for PH.

Topics: Allopurinol; Animal Feed; Animals; Antibiotics, Antineoplastic; Bleomycin; Cyclic GMP; Disease Models, Animal; Enzyme Inhibitors; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrates; Nitric Oxide Synthase Type III; Nitrites; Pulmonary Circulation; Ventricular Pressure; Xanthine Dehydrogenase

The nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) signal-transduction pathway is impaired in many cardiovascular diseases, including pulmonary arterial hypertension (PAH). Riociguat (BAY 63-2521) is a stimulator of sGC that works both in synergy with and independently of NO to increase levels of cGMP. The aims of this study were to investigate the role of NO-sGC-cGMP signaling in a model of severe PAH and to evaluate the effects of sGC stimulation by riociguat and PDE5 inhibition by sildenafil on pulmonary hemodynamics and vascular remodeling in severe experimental PAH.. Severe angioproliferative PAH was induced in rats by combined exposure to the vascular endothelial growth factor receptor antagonist SU5416 and hypoxia (SUHx). Twenty-one days thereafter rats were randomized to receive either riociguat (10 mg/kg/day), sildenafil (50 mg/kg/day) or vehicle by oral gavage, for 14 days until the day of the terminal hemodynamic measurements. Administration of riociguat or sildenafil significantly decreased right ventricular systolic pressure (RVSP). Riociguat significantly decreased RV hypertrophy (RVH) (0.55 ± 0.02, p<0.05), increased cardiac output (60.8 ± .8 mL/minute, p<0.05) and decreased total pulmonary resistance (4.03 ± 0.3 mmHg min(-1) ml(-1) 100 g BW, p<0.05), compared with sildenafil and vehicle. Both compounds significantly decreased the RV collagen content and improved RV function, but the effects of riociguat on tricuspid annular plane systolic excursion and RV myocardial performance were significantly better than those of sildenafil (p<0.05). The proportion of occluded arteries was significantly lower in animals receiving riociguat than in those receiving vehicle (p<0.05); furthermore, the neointima/media ratio was significantly lower in those receiving riociguat than in those receiving sildenafil or vehicle (p<0.05).. Riociguat and sildenafil significantly reduced RVSP and RVH, and improved RV function compared with vehicle. Riociguat had a greater effect on hemodynamics and RVH than sildenafil.

Topics: Animals; Apoptosis; Blood Pressure; Blotting, Western; Caspase 3; Cell Proliferation; Cyclic GMP; Guanylate Cyclase; Hemodynamics; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Immunohistochemistry; Indoles; Lung; Male; Nitric Oxide Synthase Type III; Phosphodiesterase 5 Inhibitors; Piperazines; Purines; Pyrazoles; Pyrimidines; Pyrroles; Random Allocation; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Sildenafil Citrate; Soluble Guanylyl Cyclase; Sulfones; Time Factors; Treatment Outcome

Soluble guanylyl cyclase (sGC) is a heterodimer. The dimerization of the enzyme is obligatory for its function in mediating actions caused by agents that elevate cyclic guanosine monophosphate (cGMP). The present study aimed to determine whether sGC dimerization is modulated by thiol-reducing agents and whether its dimerization influences relaxations in response to nitric oxide (NO).. The dimers and monomers of sGC and cGMP-dependent protein kinase (PKG) were analysed by western blotting. The intracellular cGMP content was measured by enzyme-linked immunosorbent assay. Changes in isometric tension were determined in organ chambers. In isolated porcine coronary arteries, the protein levels of sGC dimer were decreased by the thiol reductants dithiothreitol, l-cysteine, reduced l-glutathione and tris(2-carboxyethyl) phosphine. The effect was associated with reduced cGMP elevation and attenuated relaxations in response to nitric oxide donors. The dimerization of sGC and activation of the enzyme were also decreased by dihydrolipoic acid, an endogenous thiol antioxidant. Dithiothreitol at concentrations markedly affecting the dimerization of sGC had no significant effect on the dimerization of PKG or relaxation in response to 8-Br-cGMP. Relaxation of the coronary artery in response to a NO donor was potentiated by hypoxia when sGC was partly inhibited, coincident with an increase in sGC dimer and enhanced cGMP production. These effects were prevented by dithiothreitol and tris(2-carboxyethyl) phosphine.. These results demonstrate that the dimerization of sGC is exquisitely sensitive to thiol reductants compared with that of PKG, which may provide a novel mechanism for thiol-dependent modulation of NO-mediated vasodilatation in conditions such as hypoxia.

Topics: Animals; Coronary Vessels; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dimerization; Dithiothreitol; Female; Guanylate Cyclase; Hypoxia; In Vitro Techniques; Male; Nitric Oxide; Nitroso Compounds; Protein Structure, Quaternary; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Sulfhydryl Reagents; Sus scrofa; Vasodilation

Multidrug resistance-associated protein 4 (MRP4, also known as Abcc4) regulates intracellular levels of cAMP and cGMP in arterial SMCs. Here, we report our studies of the role of MRP4 in the development and progression of pulmonary arterial hypertension (PAH), a severe vascular disease characterized by chronically elevated pulmonary artery pressure and accompanied by remodeling of the small pulmonary arteries as a prelude to right heart failure and premature death. MRP4 expression was increased in pulmonary arteries from patients with idiopathic PAH as well as in WT mice exposed to hypoxic conditions. Consistent with a pathogenic role for MRP4 in PAH, WT mice exposed to hypoxia for 3 weeks showed reversal of hypoxic pulmonary hypertension (PH) following oral administration of the MRP4 inhibitor MK571, and Mrp4-/- mice were protected from hypoxic PH. Inhibition of MRP4 in vitro was accompanied by increased intracellular cAMP and cGMP levels and PKA and PKG activities, implicating cyclic nucleotide-related signaling pathways in the mechanism underlying the protective effects of MRP4 inhibition. Our data suggest that MRP4 could represent a potential target for therapeutic intervention in PAH.

Topics: Animals; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Familial Primary Pulmonary Hypertension; Humans; Hypertension, Pulmonary; Hypoxia; Leukotriene Antagonists; Lung; Mice; Mice, Knockout; Multidrug Resistance-Associated Proteins; Muscle, Smooth, Vascular; Propionates; Pulmonary Artery; Quinolines; RNA Interference; Vasoconstriction; Vasodilation

Intermittent hypoxia (IH) resulting from sleep apnea can lead to pulmonary hypertension. IH causes oxidative stress that may limit bioavailability of the endothelium-derived vasodilator nitric oxide (NO) and thus contribute to this hypertensive response. We therefore hypothesized that increased vascular superoxide anion (O(2)(-)) generation reduces NO-dependent pulmonary vasodilation following IH. To test this hypothesis, we examined effects of the O(2)(-) scavenger tiron on vasodilatory responses to the endothelium-dependent vasodilator ionomycin and the NO donor S-nitroso-N-acetylpenicillamine in isolated lungs from hypocapnic-IH (H-IH; 3 min cycles of 5% O(2)/air flush, 7 h/day, 4 wk), eucapnic-IH (E-IH; cycles of 5% O(2), 5% CO(2)/air flush), and sham-treated (air/air cycled) rats. Next, we assessed effects of endogenous O(2)(-) on NO- and cGMP-dependent vasoreactivity and measured O(2)(-) levels using the fluorescent indicator dihydroethidium (DHE) in isolated, endothelium-disrupted small pulmonary arteries from each group. Both E-IH and H-IH augmented NO-dependent vasodilation; however, enhanced vascular smooth muscle (VSM) reactivity to NO following H-IH was masked by an effect of endogenous O(2)(-). Furthermore, H-IH and E-IH similarly increased VSM sensitivity to cGMP, but this response was independent of either O(2)(-) generation or altered arterial protein kinase G expression. Finally, both H-IH and E-IH increased arterial O(2)(-) levels, although this response was more pronounced following H-IH, and H-IH exposure resulted in greater protein tyrosine nitration indicative of increased NO scavenging by O(2)(-). We conclude that IH increases pulmonary VSM sensitivity to NO and cGMP. Furthermore, endogenous O(2)(-) limits NO-dependent vasodilation following H-IH through an apparent reduction in bioavailable NO.

Topics: 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Endothelium-Dependent Relaxing Factors; Free Radical Scavengers; Hypertrophy, Right Ventricular; Hypocapnia; Hypoxia; Ionomycin; Lung; Male; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type III; Polycythemia; Pulmonary Artery; Rats; Rats, Wistar; Reactive Oxygen Species; S-Nitroso-N-Acetylpenicillamine; Superoxides; Tyrosine; Vasodilation

We previously reported that isolated endothelium-removed bovine pulmonary arteries (BPAs) contract to hypoxia associated with removal of peroxide- and cGMP-mediated relaxation. In contrast, bovine coronary arteries (BCAs) relax to hypoxia associated with cytosolic NADPH oxidation coordinating multiple relaxing mechanisms. Since we recently found that H(2)O(2) relaxes BPAs through PKG activation by both soluble guanylate cyclase (sGC)/cGMP-dependent and cGMP-independent thiol oxidation/subunit dimerization mechanisms, we investigated if these mechanisms participate in BPA contraction and BCA relaxation to hypoxia. The contraction of BPA (precontracted with 20 mM KCl) to hypoxia was associated with decreased PKG dimerization and PKG-mediated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. In contrast, exposure of 20 mM KCl-precontracted endothelium-removed BCAs to hypoxia caused relaxation and increased dimerization and VASP phosphorylation. Depletion of sGC by organoid culture of BPAs with an oxidant of the sGC heme (10 μM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) increased aerobic force generation, decreased VASP phosphorylation, and inhibited further contraction to hypoxia and changes in VASP phosphorylation. Thiol reduction with dithiothreitol increased aerobic force in BPAs and decreased PKG dimerization, VASP phosphorylation, and the contraction to hypoxia. Furthermore, PKG-1α and sGC β(1)-subunit small interfering RNA-transfected BPAs demonstrated increased aerobic K(+) force and inhibition of further contraction to hypoxia, associated with an attenuation of H(2)O(2)-elicited relaxation and VASP phosphorylation. Thus, decreases in both a sGC/cGMP-dependent and a dimerization-dependent activation of PKG by H(2)O(2) appear to contribute to the contraction of BPAs elicited by hypoxia. In addition, stimulation of PKG activation by dimerization may be important in the relaxation of coronary arteries to hypoxia.

Topics: Animals; Blotting, Western; Cattle; Cell Adhesion Molecules; Coronary Vessels; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Activation; Guanylate Cyclase; Hydrogen Peroxide; Hypoxia; Microfilament Proteins; Oxidation-Reduction; Oxygen; Phosphoproteins; Phosphorylation; Protein Multimerization; Pulmonary Artery; Receptors, Cytoplasmic and Nuclear; RNA Interference; Soluble Guanylyl Cyclase; Sulfhydryl Reagents; Tissue Culture Techniques; Transfection; Vasoconstriction; Vasoconstrictor Agents; Vasodilation

The present study investigated the mechanism underlying the transient potentiation of vasoconstriction by hypoxia in isolated porcine coronary arteries. Isometric tension was measured in rings with or without endothelium. Hypoxia (Po(2) <30 mmHg) caused a transient further increase in tension (hypoxic augmentation) in contracted (with U46619) preparations. The hypoxic response was endothelium dependent and abolished by inhibitors of nitric oxide synthase [N(ω)-nitro-L-arginine methyl ester (L-NAME)] or soluble guanylyl cyclase (ODQ and NS2028). The addition of DETA NONOate (nitric oxide donor) in the presence of L-NAME restored the hypoxic augmentation, suggesting the involvement of the nitric oxide pathway. However, the same was not observed after incubation with 8-bromo-cyclic GMP, atrial natriuretic peptide, or isoproterenol. Assay of the cyclic GMP content showed no change upon exposure to hypoxia in preparations with and without endothelium. Incubation with protein kinase G and protein kinase A inhibitors did not inhibit the hypoxic augmentation. Thus the hypoxic augmentation is dependent on nitric oxide and soluble guanylyl cyclase but independent of cyclic GMP. The hypoxic augmentation persisted in calcium-free buffer and in the presence of nifedipine, ruling out a role for extracellular calcium influx. Hypoxia did not alter the intracellular calcium concentration, as measured by confocal fluorescence microscopy. This observation and the findings that hypoxic augmentation is enhanced by thapsigargin (sarco/endoplasmic reticulum calcium ATPase inhibitor) and inhibited by HA1077 or Y27632 (Rho kinase inhibitors) demonstrate the involvement of calcium sensitization in the phenomenon.

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Coronary Vessels; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Endothelium, Vascular; Enzyme Inhibitors; Guanylate Cyclase; Hypoxia; Microscopy, Confocal; Microscopy, Fluorescence; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type III; Receptors, Cytoplasmic and Nuclear; rho-Associated Kinases; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Soluble Guanylyl Cyclase; Swine; Vasoconstriction

Whether chronic hypoxia attenuates myocardial ischemia-reperfusion injury remains controversial because conflicting data have been reported probably due to the existence of many factors influencing the functional recovery of hearts. These factors include the differences of species, the time at which hypoxia begins, the degree of hypoxia, and so on. Regarding chronic hypoxia from birth, so far the only available data are based on findings in rabbit hearts. The purpose of this study was to describe the effect of chronic hypoxia from birth on myocardial reperfusion injury in the rat heart.. Normoxic hearts were obtained from rats housed in ambient air for 6 weeks (normoxic group); hypoxic hearts were obtained from rats housed in a hypoxic chamber (13%-14% oxygen) from birth for 6 weeks (hypoxic group). Isolated, crystalloid perfused working hearts were subjected to 30 min of global normothermic ischemia followed by 15 min of reperfusion; functional recovery was then measured in the two groups. The excretion of cyclic guanosine monophosphate (cGMP) in the coronary drainage was measured at the end of the preischemia and reperfusion periods.. The percent recovery of the left ventricular developed pressure and the first derivative of left ventricular pressure were significantly better in the hypoxic group than in the normoxic group. cGMP excretion in the coronary drainage was significantly increased during both the preischemia and reperfusion periods.. Chronic hypoxia from birth increased myocardial tolerance to ischemia-reperfusion injury with increased cGMP synthesis in the isolated heart model in rats.

Topics: Animals; Chronic Disease; Cyanosis; Cyclic GMP; Disease Models, Animal; Heart; Hypoxia; KATP Channels; Male; Myocardial Contraction; Myocardial Reperfusion Injury; Myocardium; Rats; Rats, Sprague-Dawley; Recovery of Function; Up-Regulation; Ventricular Function, Left; Ventricular Pressure

The three Drosophila atypical soluble guanylyl cyclases, Gyc-89Da, Gyc-89Db, and Gyc-88E, have been proposed to act as oxygen detectors mediating behavioral responses to hypoxia. Drosophila larvae mutant in any of these subunits were defective in their hypoxia escape response-a rapid cessation of feeding and withdrawal from their food. This response required cGMP and the cyclic nucleotide-gated ion channel, cng, but did not appear to be dependent on either of the cGMP-dependent protein kinases, dg1 and dg2. Specific activation of the Gyc-89Da neurons using channel rhodopsin showed that activation of these neurons was sufficient to trigger the escape behavior. The hypoxia escape response was restored by reintroducing either Gyc-89Da or Gyc-89Db into either Gyc-89Da or Gyc-89Db neurons in either mutation. This suggests that neurons that co-express both Gyc-89Da and Gyc-89Db subunits are primarily responsible for activating this behavior. These include sensory neurons that innervate the terminal sensory cones. Although the roles of Gyc-89Da and Gyc-89Db in the hypoxia escape behavior appeared to be identical, we also showed that changes in larval crawling behavior in response to either hypoxia or hyperoxia differed in their requirements for these two atypical sGCs, with responses to 15% oxygen requiring Gyc-89Da and responses to 19 and 25% requiring Gyc-89Db. For this behavior, the identity of the neurons appeared to be critical in determining the ability to respond appropriately.

Topics: Amino Acid Sequence; Animals; Behavior, Animal; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Down-Regulation; Drosophila melanogaster; Gene Expression Regulation, Enzymologic; Guanylate Cyclase; Hypoxia; Ion Channel Gating; Ion Channels; Larva; Molecular Sequence Data; Neurons; Oxygen; Rats; Solubility

It was investigated the intercommunication of the functional state of cyclic nucleotides (CN) system and morphofunctional states of lung and heart tissues and influence on these state of lipid peroxidation processes and adenylate cyclase system activity under development in organism of the hypoxic states of different genesis. Obtained dates testify that with the changes of cAMP and cGMP concentrations, and also with their ratio the level of hyperhydratation of air-blood and blood-tissue barriers under the used influence on the organism are closely correlated. Intercommunication of the cyclic nucleotides system functioning and tissues oxygen consumption at the used influences carries sufficiently difficult and ambiguous character. It was shown that exactly at blood loss in lung and heart tissues there were the parallel diminishing of concentrations both cAMP and cGMP. It is accepted to correlate with a favorable histological dynamics. Exactly under these conditions the correlation of CN concentrations is possible to consider balanced and sent to limitation of displays of unfavorable influence of blood loss on investigated tissues ultrastructure. So, there is a tissue specific and depending from the type of influence on the organism character of intercommunication of the functional state of cyclic nucleotides system and morphofunctional states of lung and heart tissues.

Topics: Adaptation, Physiological; Animals; Cyclic AMP; Cyclic GMP; Hemorrhage; Hypoxia; Lipid Peroxidation; Lung; Male; Myocardium; Nucleotides, Cyclic; Oxygen Consumption; Rats; Rats, Wistar

Pseudomonas aeruginosa encodes many enzymes that are potentially associated with the synthesis or degradation of the widely conserved second messenger cyclic-di-GMP (c-di-GMP). In this study, we show that mutation of rbdA, which encodes a fusion protein consisting of PAS-PAC-GGDEF-EAL multidomains, results in decreased biofilm dispersal. RbdA contains a highly conserved GGDEF domain and EAL domain, which are involved in the synthesis and degradation of c-di-GMP, respectively. However, in vivo and in vitro analyses show that the full-length RbdA protein only displays phosphodiesterase activity, causing c-di-GMP degradation. Further analysis reveals that the GGDEF domain of RbdA plays a role in activating the phosphodiesterase activity of the EAL domain in the presence of GTP. Moreover, we show that deletion of the PAS domain or substitution of the key residues implicated in sensing low-oxygen stress abrogates the functionality of RbdA. Subsequent study showed that RbdA is involved in positive regulation of bacterial motility and production of rhamnolipids, which are associated with biofilm dispersal, and in negative regulation of production of exopolysaccharides, which are required for biofilm formation. These data indicate that the c-di-GMP-degrading regulatory protein RbdA promotes biofilm dispersal through its two-pronged effects on biofilm development, i.e., downregulating biofilm formation and upregulating production of the factors associated with biofilm dispersal.

Topics: Biofilms; Cyclic GMP; Gene Expression Regulation, Bacterial; Glycolipids; Guanosine Triphosphate; Hydrolysis; Hypoxia; Locomotion; Phosphoric Diester Hydrolases; Polysaccharides, Bacterial; Protein Structure, Tertiary; Pseudomonas aeruginosa

Development of intracellular calcium overload is an important pathophysiological factor in myocardial ischemia/reperfusion or anoxia/reoxygenation injury. Recent studies have shown that Sodium Ferulate (SF) stimulates nitric oxide (NO) production and exerts a cardioprotective effect in the ischemia-reperfused heart. However, it has not been determined whether the cardioprotection of SF is associated with suppression of Ca(2+) overload via NO/cyclic GMP (cGMP)/cGMP-dependent protein kinase (PKG) pathway. In this work, after cardiomyocytes were incubated with 100, 200, 400, or 800 microM SF for 3 h, anoxia/reoxygenation injury was induced and intracellular Ca(2+) concentration, NO synthase (NOS) activity, guanylate cyclase activity, NO, and cGMP formation were measured appropriately. The results showed that treatment with SF concentration-dependently inhibited calcium overload induced by anoxia/reoxygenation. We also demonstrated that SF (100-800 microM) concentration dependently enhanced NO and cGMP formation through increasing NOS activity and guanylate cyclase activity in the cardiomyocytes. On the contrary, inhibition of calcium overload by SF was markedly attenuated by addition of an NOS inhibitor, an NO scavenger, an soluble guanylate cyclase inhibitor, and a PKG inhibitor: N(G)-nitro-l-arginine methyl ester (L-NAME, 100 microM), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide (c-PTIO, 1.0 microM), 1H-[1, 2, 4] oxadiazolo [4, 3-alpha] quinoxalin-1-one (ODQ, 20 microM) and KT5823 (0.2 microM), respectively. Our findings indicate that SF significantly attenuates anoxia/reoxygenation-induced Ca(2+) overload and improves cell survival in cultured cardiomyocytes through NO/cGMP/PKG signal pathway.

Topics: Animals; Animals, Newborn; Calcium; Coumaric Acids; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Gene Expression Regulation; Guanylate Cyclase; Hypoxia; Myocytes, Cardiac; Nitric Oxide; Nitric Oxide Synthase; Oxygen; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Solubility

This article summarizes perspectives on how reactive oxygen species (ROS) and redox signaling mechanisms participate in regulating vascular smooth muscle function that have resulted from our studies over the past 25 years in areas including oxygen sensing and the regulation of cGMP production by soluble guanylate cyclase (sGC) that were presented in the Robert M. Berne Distinguished Lectureship at the 2008 Experimental Biology Meeting. It considers mechanisms controlling the activity of sources of ROS including Nox oxidases and mitochondria by physiological stimuli, vascular diseases processes, and metabolic mechanisms linked to NAD(P)H redox and hypoxia. Metabolic interactions of individual ROS such as hydrogen peroxide with cellular peroxide metabolizing enzymes are viewed as some of the most sensitive ways of influencing cellular signaling systems. The control of cytosolic NADPH redox also seems to be a major contributor to bovine coronary arterial relaxation to hypoxia, where its oxidation functions to coordinate the lowering of intracellular calcium, whereas increased cytosolic NADPH generation in pulmonary arteries appears to maintain elevated Nox oxidase activity, and relaxation to hydrogen peroxide, which is attenuated by hypoxia. The sensitivity of sGC to nitric oxide seems to be regulated by thiol and heme redox systems controlled by cytosolic NADPH. Heme biosynthesis and metabolism are also important factors regulating the sGC system. The signaling pathways controlling oxidases and their colocalization with redox-regulated systems enables selective activation of numerous regulatory mechanisms influencing vascular function in physiological processes and the progression of aging-associated vascular diseases.

Topics: Aging; Animals; Calcium; Cyclic GMP; Guanylate Cyclase; Heme Oxygenase (Decyclizing); Humans; Hypoxia; Mitochondria, Muscle; Muscle, Smooth, Vascular; NADPH Oxidases; Nitric Oxide; Oxidation-Reduction; Reactive Oxygen Species; Signal Transduction; Vascular Diseases; Vasoconstriction; Vasodilation

A broad variety of evidence obtained largely in pulmonary vasculature suggests that chronic hypoxia modulates vasoreactivity to nitric oxide (NO). The present study explores the general hypothesis that chronic hypoxia also modulates cerebrovascular reactivity to NO, and does so by modulating the activity of soluble guanylate cyclase (sGC), the primary target for NO in vascular smooth muscle. Pregnant and nonpregnant ewes were maintained at either sea level or at 3,820 m for the final 110 days of gestation, at which time middle cerebral arteries from term fetal lambs and nonpregnant adults were harvested. In both fetal and adult arteries, NO-induced vasodilatation was attenuated by chronic hypoxia and completely inhibited by 10 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of sGC. sGC abundance (in ng sGC/mg protein) measured via Western immunoblots was approximately 10-fold greater in fetal (17.6 +/- 1.6) than adult (1.7 +/- 0.3) arteries but was not affected by chronic hypoxia. The specific activity of sGC (in pmol cGMP.microg sGC(-1).min(-1)) was similar in fetal (255 +/- 64) and adult (280 +/- 75) arteries and was inhibited by chronic hypoxia in both fetal (120 +/- 10) and adult (132 +/- 26) arteries. Rates of cGMP degradation (in pmol cGMP.mg protein(-1).min(-1)) were similar in fetal (159 +/- 59) and adult (134 +/- 36) arteries but were not significantly depressed by chronic hypoxia in either fetal (115 +/- 25) or adult (108 +/- 25) arteries. The cGMP analog 8-(p-chlorophenylthio)-cGMP was a more potent vasorelaxant in fetal (pD(2) = 4.7 +/- 0.1) than adult (pD(2) = 4.3 +/- 0.1) arteries, but its ability to promote vasodilatation was not affected by chronic hypoxia in either age group. Together, these results reveal that hypoxic inhibition of NO-induced vasodilatation is attributable largely to attenuation of the specific activity of sGC and does not involve significant changes in sGC abundance, cGMP-phosphodiesterase activity, or the vasorelaxant activity of protein kinase G.

Topics: Altitude Sickness; Animals; Cerebral Arteries; Chronic Disease; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Disease Models, Animal; Endothelium-Dependent Relaxing Factors; Enzyme Inhibitors; Female; Fetus; Guanylate Cyclase; Hypoxia; Muscle, Smooth, Vascular; Nitric Oxide; Oxadiazoles; Pregnancy; Quinoxalines; Receptors, Cytoplasmic and Nuclear; Sheep; Soluble Guanylyl Cyclase; Vasodilation

Despite considerable research attention focused on mechanisms underlying neural spreading depression (SD), because of its association with important human CNS pathologies, such as stroke and migraine, little attention has been given to explaining its occurrence and regulation in invertebrates. In the locust metathoracic ganglion (MTG), an SD-like event occurs during heat and anoxia stress, which results in cessation of neuronal output for the duration of the applied stress. SD-like events were characterized by an abrupt rise in extracellular potassium ion concentration ([K(+)](o)) from a baseline concentration of approximately 8 to >30 mm, which returned to near baseline concentrations after removal of the applied stress. After return to baseline [K(+)](o), neuronal output (ventilatory motor pattern activity) from the MTG recovered. Unlike mammalian neurons, which depolarize almost completely during SD, locust neurons only partially depolarized. SD-like events in the locust CNS were suppressed by pharmacological inhibition of the nitric oxide/cyclic guanosine monophosphate/protein kinase G (NO/cGMP/PKG) pathway and were exacerbated by its activation. Also, environmental stressors such as heat and anoxia increased production of nitric oxide in the locust CNS. Finally, for the intact animal, manipulation of the pathway affected the speed of recovery from suffocation by immersion under water. We propose that SD-like events in locusts provide an adaptive mechanism for surviving extreme environmental conditions. The highly conserved nature of the NO/cGMP/PKG signaling pathway suggests that it may be involved in modulating SD in other organisms, including mammals.

Topics: Animals; Cortical Spreading Depression; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Grasshoppers; Hot Temperature; Hypoxia; Male; Motor Activity; Neural Pathways; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Phosphodiesterase Inhibitors; Potassium; Signal Transduction; Stress, Physiological

Severe pulmonary hypertension (PH) is a disabling disease with high mortality, characterized by pulmonary vascular remodeling and right heart hypertrophy. In mice with PH induced by chronic hypoxia, we examined the acute and chronic effects of the soluble guanylate cyclase (sGC) activator HMR1766 on hemodynamics and pulmonary vascular remodeling. In isolated perfused mouse lungs from control animals, HMR1766 dose-dependently inhibited the pressor response of acute hypoxia. This dose-response curve was shifted leftward when the effects of HMR1766 were investigated in isolated lungs from chronic hypoxic animals for 21 days at 10% oxygen. Mice exposed for 21 or 35 days to chronic hypoxia developed PH, right heart hypertrophy, and pulmonary vascular remodeling. Treatment with HMR1766 (10 mg x kg(-1) x day(-1)), after full establishment of PH from day 21 to day 35, significantly reduced PH, as measured continuously by telemetry. In addition, right ventricular (RV) hypertrophy and structural remodeling of the lung vasculature were reduced. Pharmacological activation of oxidized sGC partially reverses hemodynamic and structural changes in chronic hypoxia-induced experimental PH.

Topics: Animals; Cardiomegaly; Cyclic GMP; Guanylate Cyclase; Hemodynamics; Hypertension, Pulmonary; Hypoxia; Mice; Myocytes, Smooth Muscle; ortho-Aminobenzoates; Pulmonary Artery; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Sulfonamides; Superoxides; Vasoconstriction

Statins have been proposed to be a potential treatment for pulmonary arterial hypertension. If introduced into clinical practice, the statin would have to be used in conjunction with established therapy. We investigated the effects of combining simvastatin with a phosphodiesterase type-5 inhibitor, sildenafil, in the rat model of hypoxia-induced pulmonary hypertension. Rats were allocated to either: 1) a prevention protocol, to receive simvastatin 20 mg x kg(-1) x day(-1) by intraperitoneal injection or sildenafil 75 mg x kg(-1) x day(-1) orally or the combination (or vehicle) for 2 weeks beginning at the start of exposure to hypoxia (10% inspired oxygen); or 2) a treatment protocol, where the same agents were administered in the last 2 weeks of a 4-week period of hypoxia. In both protocols, the combination of sildenafil and simvastatin lowered pulmonary artery pressure and produced a significantly greater reduction in right ventricular hypertrophy and pulmonary vascular muscularisation than either drug alone. Moreover, the combination augmented significantly endothelial nitric oxide synthase expression and cGMP levels in the lung and right ventricle above that produced by either drug independently and resulted in greater inhibition of RhoA activity. These data suggest that simvastatin can be usefully combined with sildenafil in the treatment of pulmonary arterial hypertension to achieve greater therapeutic benefit.

Topics: Animals; Cyclic GMP; Disease Models, Animal; Drug Therapy, Combination; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Male; Nitric Oxide Synthase Type III; Phosphodiesterase Inhibitors; Piperazines; Pulmonary Circulation; Purines; Rats; Rats, Sprague-Dawley; rhoA GTP-Binding Protein; Signal Transduction; Sildenafil Citrate; Simvastatin; Sulfones

Topics: Acute Disease; Animals; Calcium Signaling; Cyclic GMP; Endothelial Cells; Humans; Hypoxia; Metallothionein; Mice; Mice, Knockout; Models, Biological; Nitric Oxide; Nitrosation; Protein Kinase C; Protein Processing, Post-Translational; Pulmonary Artery; Rats; Signal Transduction; Vascular Resistance; Vasoconstriction; Zinc

Exposure to hypoxia triggers a variety of adverse effects in the brain that arise from metabolic stress and induce neuron apoptosis. Overexpression of the hypoxia-inducible factor-1alpha (HIF-1alpha) is believed to be a major candidate in orchestrating the cell's defense against stress. To test the impact of HIF-1alpha on apoptosis during chronic hypoxia in vivo, we examined the protective effect of modulating the nitric oxide (NO)/cGMP pathway by sildenafil, a selective inhibitor of phosphodiesterase-5 (PDE-5). Male ICR/CD-1 mice were divided into 3 groups (n = 6/group): normoxic (21% O(2)), hypoxic (9.5% O(2)), and hypoxic with sildenafil (1.4-mg/kg intraperitoneal injections daily). At the end of the 8-day treatment period, the mice were euthanized and cerebral cortex biopsies were harvested for analyses. We found that sildenafil: (1) did not significantly alter the hypoxia-induced weight loss and hemoglobin increase, but did augment plasma nitrates+nitrites and the tissue content of cGMP and phosphorylated (P) NO synthase III; (2) reversed the hypoxia-induced neuron apoptosis (terminal deoxynucleotidyl transferase positivity and double-staining immunofluorescence, P = 0.009), presumably through increased bcl-2/Bax (P = 0.0005); and (3) did not affect HIF-1alpha, but rather blunted the hypoxia-induced increase in P-ERK1/2 (P = 0.0002) and P-p38 (P = 0.004). We conclude that upregulating the NO/cGMP pathway by PDE-5 inhibition during hypoxia reduces neuron apoptosis, regardless of HIF-1alpha, through an interaction involving ERK1/2 and p38.

Topics: Animals; Apoptosis; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Mice, Inbred ICR; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Models, Animal; Neurons; Nitric Oxide; p38 Mitogen-Activated Protein Kinases; Phosphodiesterase 5 Inhibitors; Phosphodiesterase Inhibitors; Piperazines; Purines; Signal Transduction; Sildenafil Citrate; Sulfones; Vascular Endothelial Growth Factor A

The roles of Rho kinase (ROCK) and cGMP-dependent protein kinase (PKG) in cGMP-mediated relaxation of fetal pulmonary veins exposed to chronic hypoxia (CH) were investigated. Fourth generation pulmonary veins were dissected from near-term fetuses ( approximately 140 days of gestation) delivered from ewes exposed to chronic high altitude hypoxia for approximately 110 days (CH) and from control ewes. After constriction with endothelin-1, 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) caused a similar relaxation of both control and CH vessels. Rp-8-Br-PET-cGMPS (a PKG inhibitor) inhibited whereas Y-27632 (a ROCK inhibitor) augmented relaxation of control veins to 8-Br-cGMP. These effects were significantly diminished in CH veins. PKG protein expression and activity were greater whereas ROCK protein expression and activity were less in CH vessels compared with controls. Phosphorylation of threonine 696 (ROCK substrate) and serine 695 (PKG substrate) of the regulatory myosin phosphatase targeting subunit MYPT1 of myosin light chain (MLC) phosphatase was stimulated to a lesser extent in CH than in control veins by endothelin-1 (ROCK stimulant) and 8-Br-cGMP (PKG stimulant), respectively. The phosphorylation and dephosphorylation of MLC caused by endothelin-1 and 8-Br-cGMP, respectively, were less in CH veins than in controls. These results suggest that CH in utero upregulates PKG activity but attenuates PKG action in fetal pulmonary veins. These effects are offset by the diminished ROCK action on MYPT1 and MLC and thus lead to an unaltered response to cGMP.

Topics: Altitude; Animals; Chronic Disease; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Female; Fetus; Hypoxia; In Vitro Techniques; Models, Biological; Myosin Light Chains; Myosin-Light-Chain Kinase; Phosphorylation; Phosphoserine; Phosphothreonine; Pulmonary Veins; rho-Associated Kinases; Sheep; Vasodilation

Ischemia-reperfusion causes endothelial dysfunction. Prolongation of acidosis during initial cardiac reperfusion limits infarct size in animal models, but the effects of acidic reperfusion on vascular function are unknown. The present work analyzes the effects of acidic reoxygenation on vascular responses to different agonists in rat aortic rings. Arterial rings obtained from Sprague-Dawley rat aorta were placed in organ baths containing a Krebs solution oxygenated at 37 degrees C (pH 7.4). After equilibration (30 mN, 1 h), the effects of acidosis (pH 6.4) on aortic responses to acetylcholine and norepinephrine were initially assessed under normoxic conditions. Thereafter, the effects of acidosis during hypoxia (1 h) or reoxygenation on aortic responses to acetylcholine, norepinephrine, or sodium nitroprusside were analyzed and compared with those observed in control rings. Acidosis did not modify aortic responses to acetylcholine or adrenaline during normoxia. In contrast, rings submitted to hypoxia and reoxygenated at pH 7.4 showed a reduction in vasodilator responses to acetylcholine and in contractions to norepinephrine with no change in responses to sodium nitroprusside. Reoxygenation at pH 6.4 did not modify the depressed response to norepinephrine but enhanced the recovery of acetylcholine-induced vasorelaxation. Cumulative concentration-response curves to acetylcholine showed an increased responsiveness to this drug in rings reoxygenated at a low pH. This functional improvement was associated with the preservation of aortic cGMP content after stimulation of reoxygenated rings with acetylcholine. In conclusion, acidic reoxygenation preserves endothelial function in arterial rings submitted to simulated ischemia, likely through the preservation of cGMP signaling.

Topics: Acetylcholine; Acidosis; Animals; Aorta, Thoracic; Cell Death; Cyclic GMP; Dose-Response Relationship, Drug; Endothelium, Vascular; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Ischemia; Male; Nitroprusside; Norepinephrine; Rats; Rats, Sprague-Dawley; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

The availability of inhibitors of cGMP-specific phosphodiesterase 5 (PDE 5), such as sildenafil, has revolutionized the treatment of pulmonary hypertension (PH). Sildenafil may exert its protective effects in a mechanism-based fashion by targeting a pathophysiologically attenuated NO-cGMP signaling pathway. To elucidate this, we analyzed changes in the pulmonary expression and activity of key enzymes of NO-cGMP signaling as well as the functional pulmonary responses to sildenafil in the 5 or 21 day hypoxia mouse model of PH. Surprisingly, we found doubled NO synthase (NOS) II and III levels, no evidence for attenuated NO bioavailability as evidenced by the nitrosative/oxidative stress marker protein nitro tyrosine, and no changes in the expression and activity of the NO receptor, soluble guanylyl cyclase (sGC). PDE 5 was either unchanged at day 5 or, after 21 days of hypoxia, even significantly decreased along with unchanged activity. Biochemically, these changes were mirrored by increased cGMP spillover into the lung perfusate and cGMP-dependent phosphorylation of the vasodilator-stimulated phosphoprotein, VASP. Sildenafil further augmented cGMP and phospho-VASP levels in lungs of mice exposed for 5 or 21 days and decreased pulmonary arterial pressure in mice after 5 days but not 21 days of hypoxia. In conclusion, NO-cGMP signaling is compensatorily up-regulated in the hypoxic mouse model of PH, and sildenafil further augments this pathway to functionally alleviate pulmonary vasoconstriction.

Topics: Animals; Cyclic GMP; Hypertension, Pulmonary; Hypoxia; Lung; Male; Mice; Mice, Inbred C57BL; Nitric Oxide; Phosphodiesterase Inhibitors; Phosphorylation; Piperazines; Purines; Signal Transduction; Sildenafil Citrate; Sulfones; Up-Regulation

Perinatal adverse events such as limitation of nutrients or oxygen supply are associated with the occurrence of diseases in adulthood, like cardiovascular diseases and diabetes. We investigated the long-term effects of perinatal hypoxia on the lung circulation, with particular attention to the nitric oxide (NO)/cGMP pathway. Mice were placed under hypoxia in utero 5 days before delivery and for 5 days after birth. Pups were then bred in normoxia until adulthood. Adults born in hypoxia displayed an altered regulation of pulmonary vascular tone with higher right ventricular pressure in normoxia and increased sensitivity to acute hypoxia compared with controls. Perinatal hypoxia dramatically decreased endothelium-dependent relaxation induced by ACh in adult pulmonary arteries (PAs) but did not influence NO-mediated endothelium-independent relaxation. The M(3) muscarinic receptor was implicated in the relaxing action of ACh and M(1) muscarinic receptor (M(1)AChR) in its vasoconstrictive effects. Pirenzepine or telenzepine, two preferential inhibitors of M(1)AChR, abolished the adverse effects of perinatal hypoxia on ACh-induced relaxation. M(1)AChR mRNA expression was increased in lungs and PAs of mice born in hypoxia. The phosphodiesterase 1 (PDE1) inhibitor vinpocetine also reversed the decrease in ACh-induced relaxation following perinatal hypoxia, suggesting that M(1)AChR-mediated alteration of ACh-induced relaxation is due to the activation of calcium-dependent PDE1. Therefore, perinatal hypoxia leads to an altered pulmonary circulation in adulthood with vascular dysfunction characterized by impaired endothelium-dependent relaxation and M(1)AChR plays a predominant role. This raises the possibility that muscarinic receptors could be key determinants in pulmonary vascular diseases in relation to "perinatal imprinting."

Topics: Acetylcholine; Animals; Cyclic GMP; Endothelium, Vascular; Female; Gene Expression Regulation; Hypoxia; Lung; Lung Diseases; Male; Mice; Nitric Oxide; Phosphodiesterase I; Pregnancy; Prenatal Exposure Delayed Effects; Pulmonary Circulation; Receptor, Muscarinic M1; Vasodilation; Vasodilator Agents

An increase in Rho kinase (ROCK) activity is implicated in chronic hypoxia-induced pulmonary hypertension. In the present study, we determined the role of ROCKs in cGMP-dependent protein kinase (PKG)-mediated pulmonary vasodilation of fetal lambs exposed to chronic hypoxia. Fourth generation pulmonary arteries were isolated from near-term fetuses ( approximately 140 days of gestation) delivered from ewes exposed to chronic high altitude hypoxia for approximately 110 days and from control ewes. In vessels constricted to endothelin-1, 8-bromoguanosine-cGMP (8-Br-cGMP) caused a smaller relaxation in chronically hypoxic (CH) vessels compared with controls. Rp-8-Br-PET-cGMPS, a PKG inhibitor, attenuated relaxation to 8-Br-cGMP in control vessels to a greater extent than in CH vessels. Y-27632, a ROCK inhibitor, significantly potentiated 8-Br-cGMP-induced relaxation of CH vessels and had only a minor effect in control vessels. The expression of PKG was increased but was not accompanied with an increase in the activity of the enzyme in CH vessels. The expression of type II ROCK and activity of ROCKs were increased in CH vessels. The phosphorylation of threonine (Thr)696 and Thr850 of the regulatory subunit MYPT1 of myosin light chain phosphatase was inhibited by 8-Br-cGMP to a lesser extent in CH vessels than in controls. The difference was eliminated by Y-27632. These results suggest that chronic hypoxia in utero attenuates PKG-mediated relaxation in pulmonary arteries, partly due to inhibition of PKG activity and partly due to enhanced ROCK activity. Increased ROCK activity may inhibit PKG action through increased phosphorylation of MYPT1 at Thr696 and Thr850.

Topics: Amides; Animals; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Female; Fetus; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Hypoxia; Intracellular Signaling Peptides and Proteins; Male; Muscle, Smooth, Vascular; Myosin-Light-Chain Phosphatase; Oxygen; Pregnancy; Protein Serine-Threonine Kinases; Pulmonary Artery; Pulmonary Circulation; Pyridines; rho-Associated Kinases; RNA, Messenger; Sheep; Vasodilation

The vasodilator adrenomedullin (AM) is up-regulated in pulmonary hypertension, and inhaled AM is beneficial in patients. Therefore, we investigated the effects of AM on pulmonary endothelin-1 (ET-1). In normoxic isolated rat lungs (IRL) and rat pulmonary artery endothelial cells (RPAEC), the calcitonin gene-related peptide type-1 receptor (CGRP1R) antagonist human (h)CGRP(8-37) decreased ET-1 secretion, and the AM receptor antagonist hAM(22-52) had no effect. Exogenous AM (1 and 10 pM) increased ET-1 levels, which was abolished by hCGRP(8-37) and protein kinase A (PKA) inhibition. At 50 and 100 pM, AM decreased ET-1, an effect sensitive to hAM(22-52), NO inhibition, and protein kinase G (PKG) inhibition. In RPAEC, these results were attributed to altered ET-1 gene expression; low exogenous AM also promoted activity of endothelin-converting enzyme, and high AM increased the number of endothelin type-B (ETB) receptor sites. Hypoxia significantly elevated AM and ET-1 levels in IRL and RPAEC, and hAM(22-52), NO inhibition, or PKG inhibitors caused a further ET-1 rise. These interventions also prevented the hypoxia-related increase in ETB sites in RPAEC. In RPAEC, both high AM and hypoxia down-regulated receptor activity-modifying protein (RAMP)1, but they up-regulated RAMP2 protein and AM receptor sites, and RAMP2 silencing by small interference RNA proved its pivotal role for signal switching. In conclusion, endogenous pulmonary AM up-regulates ET-1 and endothelin-converting enzyme activity under physiological conditions, via CGRP1R and PKA. In contrast, hypoxia-induced high AM levels, via AM1 receptor and NO/PKG, down-regulate ET-1 gene expression and promote expression of ETB receptors. This hypoxia-related switch of AM signaling can be attributed to up-regulation of the RAMP2/AM1 receptor system.

Topics: Adrenomedullin; Animals; Anti-Inflammatory Agents, Non-Steroidal; Blotting, Western; Calcitonin Gene-Related Peptide; Cyclic AMP; Cyclic GMP; Endothelial Cells; Endothelin-1; Hypoxia; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Lung; Male; Membrane Proteins; Nitric Oxide; Rats; Rats, Wistar; Receptor Activity-Modifying Protein 2; Receptor Activity-Modifying Proteins; Receptor, Endothelin B; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Solvents

It has been reported that dehydroepiandrosterone is a pulmonary vasodilator and inhibits chronic hypoxia-induced pulmonary hypertension. Additionally, dehydroepiandrosterone has been shown to improve systemic vascular endothelial function. Thus, we hypothesized that chronic treatment with dehydroepiandrosterone would attenuate hypoxic pulmonary hypertension by enhancing pulmonary artery endothelial function.. Rats were randomly assigned to five groups. Three groups received food containing 0, 0.3, or 1% dehydroepiandrosterone during a 3-wk-exposure to simulated high altitude (HA). The other 2 groups were kept at Denver's low altitude (LA) and received food containing 0 or 1% dehydroepiandrosterone. Dehydroepiandrosterone dose-dependently inhibited hypoxic pulmonary hypertension (mean pulmonary artery pressures after treatment with 0, 0.3, and 1% dehydroepiandrosterone=45+/-5, 33+/-2*, and 25+/-1*# mmHg, respectively. *P<0.05 vs. 0% and # vs. 0.3%). Dehydroepiandrosterone (1%, 3 wks) treatment started after rats had been exposed to 3-wk hypoxia also effectively reversed established hypoxic pulmonary hypertension. Pulmonary artery rings isolated from both LA and HA rats treated with 1% dehydroepiandrosterone showed enhanced relaxations to acetylcholine and sodium nitroprusside, but not to 8-bromo-cGMP. In the pulmonary artery tissue from dehydroepiandrosterone-treated LA and HA rats, soluble guanylate cyclase, but not endothelial nitric oxide synthase, protein levels were increased.. These results indicate that the protective effect of dehydroepiandrosterone against hypoxic pulmonary hypertension may involve upregulation of pulmonary artery soluble guanylate cyclase protein expression and augmented pulmonary artery vasodilator responsiveness to nitric oxide.

Topics: Acetylcholine; Animals; Blotting, Western; Cyclic GMP; Dehydroepiandrosterone; Dehydroepiandrosterone Sulfate; Dose-Response Relationship, Drug; Drug Administration Schedule; Estradiol; Guanylate Cyclase; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Lung; Male; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase Type III; Nitroprusside; Pulmonary Artery; Random Allocation; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Testosterone; Up-Regulation; Vasodilator Agents

The Western painted turtle survives months without oxygen. A key adaptation is a coordinated reduction of cellular ATP production and utilization that may be signaled by changes in the concentrations of reactive oxygen species (ROS) and cyclic nucleotides (cAMP and cGMP). Little is known about the involvement of cyclic nucleotides in the turtle's metabolic arrest and ROS have not been previously measured in any facultative anaerobes. The present study was designed to measure changes in these second messengers in the anoxic turtle. ROS were measured in isolated turtle brain sheets during a 40-min normoxic to anoxic transition. Changes in cAMP and cGMP were determined in turtle brain, pectoralis muscle, heart and liver throughout 4 h of forced submergence at 20-22 degrees C. Turtle brain ROS production decreased 25% within 10 min of cyanide or N(2)-induced anoxia and returned to control levels upon reoxygenation. Inhibition of electron transfer from ubiquinol to complex III caused a smaller decrease in [ROS]. Conversely, inhibition of complex I increased [ROS] 15% above controls. In brain [cAMP] decreased 63%. In liver [cAMP] doubled after 2 h of anoxia before returning to control levels with prolonged anoxia. Conversely, skeletal muscle and heart [cAMP] remained unchanged; however, skeletal muscle [cGMP] became elevated sixfold after 4 h of submergence. In liver and heart [cGMP] rose 41 and 127%, respectively, after 2 h of anoxia. Brain [cGMP] did not change significantly during 4 h of submergence. We conclude that turtle brain ROS production occurs primarily between mitochondrial complexes I and III and decreases during anoxia. Also, cyclic nucleotide concentrations change in a manner suggestive of a role in metabolic suppression in the brain and a role in increasing liver glycogenolysis.

Topics: Adaptation, Physiological; Animals; Cerebral Cortex; Cyclic AMP; Cyclic GMP; Glycogenolysis; Hypoxia; Liver; Oxygen; Reactive Oxygen Species; Signal Transduction; Turtles

We examined the effects of acute hypoxia on vascular tone and coronary blood flow (CBF) in rabbit coronary arteries. In the pressurized arterial preparation of small arteries (<100 mum) and the Langendorff-perfused rabbit hearts, hypoxia induced coronary vasodilation and increased CBF in the presence of glibenclamide (K(ATP) channel blocker), Rp-8-Br-PET-cGMPs [cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibitor, Rp-cGMPs], and methionyl transfer RNA synthetase (MRS) 1334 (adenosine A(3) receptor inhibitor); these increases were inhibited by the inward rectifier K(+) (Kir) channel inhibitor, Ba(2+). These effects were blocked by the adenylyl cyclase inhibitor SQ 22536 and by the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) inhibitors Rp-8-CPT-cAMPs (Rp-cAMPs) and KT 5720. However, cGMP-dependent protein kinase was not involved in the hypoxia-induced increases of the vascular diameter and CBF. In summary, our results suggest that acute hypoxia can induce the opening of Kir channels in coronary artery that has small diameter (<100 mum) by activating the cAMP and PKA signalling pathway, which could contribute to vasodilation and, therefore, increased CBF.

Topics: Acute Disease; Adenine; Animals; Blood Pressure; Carbazoles; Coronary Circulation; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Female; Glyburide; Hypoxia; In Vitro Techniques; Indoles; Male; Potassium Channel Blockers; Potassium Channels, Inwardly Rectifying; Pyrroles; Rabbits; Signal Transduction; Thionucleotides; Vasodilation

Nitric oxide (NO) activates soluble guanylate cyclase (sGC), a heterodimer composed of alpha- and beta-subunits, to produce cGMP. NO reduces pulmonary vascular remodeling, but the role of sGC in vascular responses to acute and chronic hypoxia remains incompletely elucidated. We therefore studied pulmonary vascular responses to acute and chronic hypoxia in wild-type (WT) mice and mice with a nonfunctional alpha1-subunit (sGCalpha1-/-).. sGCalpha1-/- mice had significantly reduced lung sGC activity and vasodilator-stimulated phosphoprotein phosphorylation. Right ventricular systolic pressure did not differ between genotypes at baseline and increased similarly in WT (22+/-2 to 34+/-2 mm Hg) and sGCalpha1-/- (23+/-2 to 34+/-1 mm Hg) mice in response to acute hypoxia. Inhaled NO (40 ppm) blunted the increase in right ventricular systolic pressure in WT mice (22+/-2 to 24+/-2 mm Hg, P<0.01 versus hypoxia without NO) but not in sGCalpha1-/- mice (22+/-1 to 33+/-1 mm Hg) and was accompanied by a significant rise in lung cGMP content only in WT mice. In contrast, the NO-donor sodium nitroprusside (1.5 mg/kg) decreased systemic blood pressure similarly in awake WT and sGCalpha1-/- mice as measured by telemetry (-37+/-2 versus -42+/-4 mm Hg). After 3 weeks of hypoxia, the increases in right ventricular systolic pressure, right ventricular hypertrophy, and muscularization of intra-acinar pulmonary vessels were 43%, 135%, and 46% greater, respectively, in sGCalpha1-/- than in WT mice (P<0.01). Increased remodeling in sGCalpha1-/- mice was associated with an increased frequency of 5'-bromo-deoxyuridine-positive vessels after 1 and 3 weeks (P<0.01 versus WT).. Deficiency of sGCalpha1 does not alter hypoxic pulmonary vasoconstriction. sGCalpha1 is essential for NO-mediated pulmonary vasodilation and limits chronic hypoxia-induced pulmonary vascular remodeling.

Topics: Acute Disease; Animals; Antimetabolites; Blood Pressure; Bromodeoxyuridine; Chronic Disease; Cyclic GMP; Dimerization; Female; Guanylate Cyclase; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Male; Mice; Mice, Mutant Strains; Nitric Oxide; Pulmonary Artery; Pulmonary Circulation; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Vasodilation; Ventricular Function, Right

The present study tests the hypothesis that chronic hypoxia enhances reactivity to nitric oxide (NO) through age-dependent increases in soluble guanylate cyclase (sGC) and protein kinase G (PKG) activity. In term fetal and adult ovine carotids, chronic hypoxia had no significant effect on mRNA levels for the beta1-subunit of sGC, but depressed sGC abundance by 16% in fetal and 50% in adult arteries, through possible depression of rates of mRNA translation (15% in fetal and 50% in adult) and/or increased protein turnover. Chronic hypoxia also depressed the catalytic activity of sGC, but only in fetal arteries (63%). Total sGC activity was reduced by chronic hypoxia in both fetal (69%) and adult (37%) carotid homogenates, but this effect was not observed in intact arteries when sGC activity was measured by timed accumulation of cGMP. In intact arteries treated with 300 microM 3-isobutyl-1-methylxanthine (IBMX), chronic hypoxia dramatically enhanced sGC activity in fetal (186%) but not adult (89%) arteries. This latter observation suggests that homogenization either removed an sGC activator, released an sGC inhibitor, or altered the phosphorylation state of the enzyme, resulting in reduced activity. In the absence of IBMX, chronic hypoxia had no significant effect on rates of cGMP accumulation. Chronic hypoxia also depressed the ability of the cGMP analog, 8-(p-chlorophenylthio)-cGMP, to promote vasorelaxation in both fetal (8%) and adult (12%) arteries. Together, these results emphasize the fact that intact and homogenized artery studies of sGC activity do not always yield equivalent results. The results further suggest that enhancement of reactivity to NO by chronic hypoxia must occur upstream of PKG and can only be possible if changes in cGMP occurred in functional compartments that afforded either temporal or chemical protection to the actions of phosphodiesterase. The range and age dependence of hypoxic effects observed also suggest that some responses to hypoxia must be compensatory and homeostatic, with reactivity to NO as the primary regulated variable.

Topics: 1-Methyl-3-isobutylxanthine; Aging; Animals; Carotid Arteries; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Enzyme Activation; Female; Guanylate Cyclase; Hypoxia; Nitric Oxide; Phosphodiesterase Inhibitors; Pregnancy; Pregnancy, Animal; RNA, Messenger; Sheep; Thionucleotides

Carbon monoxide (CO), an endogenous cytoprotective product of heme oxygenase type-1 regulates target thrombotic and inflammatory genes in ischemic stress. Regulation of the gene encoding early growth response 1 (Egr-1), a potent transcriptional activator of deleterious thrombotic and inflammatory cascades, may govern CO-mediated ischemic lung protection. The exact signaling mechanisms underlying CO-mediated cytoprotection are not well understood. In this study we tested the hypothesis that inhibition of mitogen-activated protein kinase-dependent Egr-1 expression may be pivotal in CO-mediated ischemic protection. In an in vivo isogeneic rat lung ischemic injury model, inhaled CO not only diminished fibrin accumulation and leukostasis and improved gas exchange and survival but also suppressed extracellular signal-regulated kinase (ERK) activation, Egr-1 expression, and Erg DNA-binding activity in lung tissue. Additionally, CO-mediated inhibition of Egr-1 reduced expression of target genes, such as tissue factor, serpine-1, interleukin-1, and TNF-alpha. However, CO failed to inhibit serpine-1 expression after unilateral lung ischemia in mice null for the Egr-1 gene. In RAW macrophages in vitro, hypoxia-induced Egr-1 mRNA expression was ERK-dependent, and CO-mediated suppression of ERK activation resulted in Egr-1 inhibition. Furthermore, CO suppression of ERK phosphorylation was reversed by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one but was insensitive to cAMP-dependent protein kinase A inhibition with H89 and NO synthase inhibition with l-nitroarginine methyl ester. This finding indicates that CO suppresses ERK in a cGMP-dependent but cAMP/protein kinase A- and NO-independent manner. Together, these data identify a unifying molecular mechanism by which CO interrupts proinflammatory and prothrombotic mediators of ischemic injury.

Topics: Animals; Carbon Monoxide; Cell Line; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Early Growth Response Protein 1; Gene Expression Regulation; Graft Survival; Hypoxia; Ischemia; Lung Diseases; Lung Transplantation; Male; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; Nitric Oxide; Rats

To determine the effects of sildenafil citrate on fetal growth in maternal rats exposed to hypoxia.. Timed pregnant rats were randomized to either hypoxia or control on gestational days (GD) 18-20, and received either sildenafil (45 mg/kg) orally every 12 h on GD 18-21 or an equal volume of sterile water. Fetal pups were retrieved by laparotomy on GD 21. Pup weight and length were evaluated and cGMP measured in maternal and fetal blood.. In the non-hypoxic rats, sildenafil exposure was associated with a decrease in size(4.75 +/- 0.43 vs. 5.11 +/- 0.34 g, p = 0.00). In contrast, in the hypoxic rat model, sildenafil exposure was associated with increased size of the offspring (5.48 +/- 0.45 vs. 5.16 +/- 0.36 g, p = 0.016). Maternal cGMP levels were increased in the presence of both sildenafil and hypoxia (23.0 +/- 10.5 vs. 15.6 +/- 2.7 pmol/ml, p = 0.001).. Exposure to sildenafil in a non-hypoxic setting results in a decrease in fetal size. Sildenafil in the presence of a stimulus, hypoxia, will lead to increased fetal size. These results suggest that sildenafil may have some influence on fetal growth. How these effects occur and by what mechanism remain to be determined.

Topics: Animals; Cyclic GMP; Female; Fetal Development; Fetal Weight; Gestational Age; Hypoxia; Piperazines; Pregnancy; Pregnancy Complications; Purines; Rats; Sildenafil Citrate; Sulfones

Devising therapies that might prevent the onset or progression of pulmonary hypertension in newborns has received little attention. Our major objective was to determine whether sildenafil, a selective phosphodiesterase inhibitor, prevents the development of an early stage of chronic hypoxia-induced pulmonary hypertension in newborn pigs. Another objective was to determine whether sildenafil causes pulmonary vasodilation without systemic vasodilation in piglets with chronic pulmonary hypertension. Piglets were raised in room air (control, n = 5) or 10-11% O(2) (hypoxic, n = 17) for 3 days. Some piglets (n = 4) received oral sildenafil, 12 mg/kg/day, throughout exposure to hypoxia. All piglets were anesthetized and catheterized, and pulmonary arterial pressure (Ppa), pulmonary wedge pressure (Pw), aortic pressure (Ao), and cardiac output (CO) were measured. Then for some piglets raised in hypoxia for 3 days, a single oral sildenafil dose (3 mg/kg, n = 6) or placebo (n = 5) was given, and hemodynamic measurements were repeated. For piglets raised in hypoxia for 3 days, mean Ppa and calculated PVR were elevated above respective values in control piglets. Mean Ppa and PVR did not differ between piglets that received sildenafil throughout exposure to hypoxia and those that did not. For piglets with chronic hypoxia-induced pulmonary hypertension that received a single oral dose of sildenafil, mean Ppa and PVR decreased, while mean Pw, CO, mean Ao, and systemic vascular resistance remained the same. All hemodynamic measurements were unchanged after placebo. Oral sildenafil did not influence the early stage of chronic hypoxia-induced pulmonary hypertension in newborn piglets. However, a single oral dose of sildenafil caused pulmonary vasodilation, without systemic vasodilation, in piglets with chronic hypoxia-induced pulmonary hypertension, which may have therapeutic implications.

Topics: Administration, Oral; Animals; Animals, Newborn; Chronic Disease; Cyclic GMP; Disease Models, Animal; Disease Progression; Dose-Response Relationship, Drug; Hemodynamics; Humans; Hypertension, Pulmonary; Hypoxia; Infant, Newborn; Infant, Newborn, Diseases; Lung; Piperazines; Pulmonary Artery; Purines; Reference Values; Sildenafil Citrate; Sulfones; Swine; Treatment Outcome; Vasodilator Agents

The potent vasomodulator adenosine (AD), thanks to the interaction with by A(1) and A(2) receptors, dilates systemic, coronary and cerebral vasculatures but exert a constrictor action in several vessels of respiratory organs. Recent investigations suggest that nitric oxide (NO) contributes to AD effects. In fish, both NO and AD induce atypical effects compared to mammals. Since there is very little information on the role of NO and its involvement in mediating the actions of AD in fish, we have analysed this question in the branchial vasculature of the elasmobranch Squalus acanthias and the teleost Anguilla anguilla using an isolated perfused head and a branchial basket preparation, respectively. In both dogfish and eel, AD dose-response curves showed a biphasic effect: vasoconstriction (pico to nanomolar range) and vasodilation (micromolar range). Both effects were abolished by the classic xanthine inhibitor theophylline (Theo) and also by specific antagonists of A(1) and A(2) receptor subtypes. To analyse the involvement of the NO/cGMP system in the AD responses, we tested a NOS inhibitor, l-NIO, and a specific soluble guanylate cyclase (sGC) blocker, ODQ. In both dogfish and eel preparations l-NIO abrogated all vasomotor effects of AD, whereas ODQ blocked the AD-mediated vasoconstriction without affecting the vasorelaxant response. This indicates that only AD-induced vasoconstriction is mediated by a NO-cGMP-dependent mechanism. By using the NO donor SIN-1, we showed a dose-dependent vasoconstrictory effect which was completely blocked by ODQ. These results provide compelling evidence that the vasoactive role of AD in the branchial circulation of S. acanthias and A. anguilla involves a NO signalling.

Topics: Adenosine; Anguilla; Animals; Cyclic GMP; Dose-Response Relationship, Drug; Female; Hypoxia; In Vitro Techniques; Male; Molsidomine; Nitric Oxide; Nitric Oxide Donors; Perfusion; Pressure; Signal Transduction; Squalus acanthias; Time Factors; Vasodilation

The activation of ATP-sensitive potassium (K ATP) ion channels in the heart is thought to exert a cardioprotective effect under low oxygen conditions, possibly enhancing tolerance of environmental hypoxia in aquatic vertebrates. The purpose of this study was to examine the possibility that hypoxia-induced activation of cardiac K ATP channels, whether in the sarcolemma (sarcK ATP) or mitochondria (mitoK ATP), enhances viability in cardiac muscle cells from a species highly tolerant of low oxygen environments, the goldfish Carassius auratus. During moderate hypoxia (6-7 kPa), the activation of sarcK ATP channels was indicated by a reduction in transmembrane action potential duration (APD). This response to hypoxia was mimicked by the NO-donor SNAP (100 micromol l(-1)) and the stable cGMP analog 8-Br-cGMP, but abolished by glibenclamide or l-NAME, an inhibitor of NO synthesis. The mitoK ATP channel opener diazoxide did not affect APD. Isolated ventricular muscle cells were then incubated under normoxic and hypoxic conditions. Cell viability was decreased in hypoxia; however, the negative effects of low oxygen were reduced during simultaneous exposure to SNAP, 8-Br-cGMP, and diazoxide. The cardioprotective effect of diazoxide, but not 8-Br-cGMP, was reduced by the mitoK ATP channel blocker 5-HD. These data suggest that hypoxia-induced activation of sarcK ATP or mitoK ATP channels could enhance tolerance of low-oxygen environments in this species, and that sarcK ATP activity is increased through a NO and cGMP-dependent pathway.

Topics: Acclimatization; Action Potentials; Analysis of Variance; Animals; Cyclic GMP; Diazoxide; Glyburide; Goldfish; Hypoxia; Mitochondria; Models, Biological; Myocardium; NG-Nitroarginine Methyl Ester; Penicillamine; Potassium Channels; Sarcolemma

We recently reported that proteinase-activated receptors type I (PAR-1) are coupled to both negative and positive invasion pathways in colonic and kidney cancer cells cultured on collagen type I gels. Here, we found that treatments with the cell-permeant analog 8-Br-cGMP and the soluble guanylate cyclase activator BAY41-2272, and Rho kinase (ROK) inhibition by Y27632 or a dominant negative form of ROK lead to PAR-1-mediated invasion through differential Rac1 and Cdc42 signaling. Hypoxia or the counteradhesive matricellular protein SPARC/BM-40 (SPARC: secreted protein acidic rich in cysteine) overexpressed during cancer progression also commutated PAR-1 to cellular invasion through the cGMP/protein kinase G (PKG) cascade, RhoA inactivation, and Rac1-dependent or -independent signaling. Cultured primary cancer cells isolated from peritoneal and pleural effusions from patients with colon cancer or other malignant tumors harbored PAR-1, as shown by RT-PCR and FACS analyses. These malignant effusions also contained high levels of activated thrombin and fibrin, and induced a proinvasive response in HCT8/S11 human colorectal cancer cells. Our data underline the essential role of the tumor microenvironment and of several commutators targeting cGMP/PKG signaling and the RhoA-ROK axis in the control of PAR-1 proinvasive activity and metastatic potential of cancer cells in distant organs and peritoneal or pleural cavities. We also add new insights into the mechanisms linking the coagulation mediators thrombin and PAR-1 in the context of blood coagulation disorders and venous thrombosis often observed in cancer patients, as described in 1865 by Armand Trousseau.

Topics: Aged; Aged, 80 and over; Animals; Cyclic GMP; Dogs; G-Protein-Coupled Receptor Kinase 1; Guanylate Cyclase; HCT116 Cells; HT29 Cells; Humans; Hypoxia; Intracellular Signaling Peptides and Proteins; Middle Aged; Neoplasm Invasiveness; Neoplasms; Osteonectin; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Receptor, PAR-1; Receptors, Antigen; Receptors, Cytoplasmic and Nuclear; rho GTP-Binding Proteins; rho-Associated Kinases; rhoA GTP-Binding Protein; Signal Transduction; Soluble Guanylyl Cyclase; Thrombin

We investigated the effects of fluvastatin on hypoxia-induced (1 to 3 weeks, 10% O2) pulmonary hypertension with focus on endothelial nitric oxide synthase (eNOS) activity.. Oral fluvastatin treatment (1 mg/kg daily) prevented the causing and progression of pulmonary hypertension as determined by the right ventricular pressure, right ventricular hypertrophy, and muscularization of pulmonary artery. We also revealed that fluvastatin treatments prevented the hypoxia-induced decrease in cGMP production in the rat lung and restored the endothelium-dependent relaxation in the pulmonary artery. We revealed that this beneficial effect was not dependent on the increase in eNOS mRNA or protein expression, but was dependent on the inhibition of the eNOS-tight coupling with caveolin-1, the eNOS dissociation from heat shock protein 90, and the decrease in eNOS Ser1177-phosphorylation induced by hypoxia. Furthermore, in a whole-mount immunostaining the hypoxia-induced eNOS protein condensation with caveolin-1 of pulmonary endothelial cells was restored by the fluvastatin-treatment.. These results suggest that the fluvastatin exerts beneficial effects on chronic hypoxia-induced pulmonary hypertension by protecting against the eNOS activity at the post-transcriptional level.

Topics: Animals; Blood Pressure; Caveolin 1; Chronic Disease; Cyclic GMP; Electrocardiography; Endothelium, Vascular; Fatty Acids, Monounsaturated; Fluvastatin; HSP90 Heat-Shock Proteins; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypertension, Pulmonary; Hypoxia; Indoles; Myocardium; Nitric Oxide Synthase Type III; Organ Size; Phosphorylation; Proto-Oncogene Proteins c-akt; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Vasodilation; Ventricular Function, Right

Tumour hypoxia is associated with resistance to therapy and with increased invasion and metastatic potential. Recent studies in our laboratory have shown that the hypoxic up-regulation of tumour cell invasiveness and chemoresistance is in part due to reduced nitric oxide (NO) signaling. Using B16F10 murine melanoma cells, we demonstrate here that the increased metastatic potential associated with exposure to hypoxia is mediated by a reduction in cGMP-dependent NO-signaling. Pre-incubation of B16F10 cells in hypoxia (1% vs. 20% O(2)) for 12 hr increased lung colonization ability by over 4-fold. This effect of hypoxia on metastasis was inhibited by co-incubation with low concentrations of the NO-mimetic drugs glyceryl trinitrate (GTN) and diethylenetriamine NO adduct (DETA/NO). In a manner similar to hypoxia, pharmacological inhibition of NO synthesis resulted in a significant increase in lung nodule formation, an effect that was prevented by co-incubation with GTN. An important NO-signaling pathway involves the activation of soluble guanylyl cyclase and the consequential generation of cGMP. Culture in the presence of a non-hydrolysable cGMP analogue (8-Br-cGMP) abrogated the hypoxia-induced lung nodule formation, suggesting that the effects of NO on metastasis are mediated via a cGMP-dependent pathway. These findings suggest that a novel mechanism whereby hypoxia regulates metastatic potential involves a downstream inhibition of cGMP-dependent NO signaling.

Topics: Animals; Cyclic GMP; Female; Hypoxia; Lung Neoplasms; Melanoma, Experimental; Mice; Neoplasm Metastasis; Neoplasm Transplantation; Nitric Oxide; Signal Transduction; Tumor Cells, Cultured; Tumor Stem Cell Assay

Differential screening of a Littorina littorea (the common periwinkle) cDNA library identified ferritin heavy chain as an anoxia-induced gene in hepatopancreas. Northern blots showed that ferritin heavy chain transcript levels were elevated twofold during anoxia exposure, although nuclear run-off assays demonstrated that ferritin heavy chain mRNAs were not transcriptionally upregulated during anoxia. Polysome analysis indicated that existing ferritin transcripts were actively translated during the anoxic period. This result was confirmed via western blotting, which demonstrated a twofold increase in ferritin heavy chain protein levels during anoxia, with a subsequent decrease to control levels during normoxic recovery. Organ culture experiments using hepatopancreas slices demonstrated a >50% increase in ferritin heavy chain transcript levels in vitro under conditions of anoxia and freezing, as well as after incubation with the second messenger cGMP. Taken together, these results suggest that ferritin heavy chain is actively regulated during anoxia exposure in the marine snail, L. littorea.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Blotting, Western; Cyclic GMP; DNA, Complementary; Ferritins; Gene Expression Regulation; Hepatopancreas; Hypoxia; Molecular Sequence Data; Polyribosomes; Protein Biosynthesis; Second Messenger Systems; Sequence Alignment; Sequence Analysis, DNA; Snails; Spectrophotometry

Erythropoietin (EPO), a hypoxia-inducible cytokine, is required for survival, proliferation, and differentiation of erythroid progenitor cells. EPO can also stimulate proliferation and angiogenesis of endothelial cells that express EPO receptors (EPORs). In this study we investigated the EPO response of vascular endothelial cells at reduced oxygen tension (5% and 2%), in particular the effect of EPO on nitric oxide (NO) release. Endothelial nitric oxide synthase (eNOS) produces NO, which maintains blood pressure homeostasis and blood flow. We find that EPOR is inducible by EPO in primary human endothelial cells of vein (HUVECs) and artery (HUAECs) and cells from a human bone marrow microvascular endothelial line (TrHBMEC) to a much greater extent at low oxygen tension than in room air. We found a corresponding increase in eNOS expression and NO production in response to EPO during hypoxia. Stimulation of NO production was dose dependent on EPO concentration and was maximal at 5 U/mL. NO activates soluble guanosine cyclase to produce cyclic guanosine monophosphate (cGMP), and we observed that EPO induced cGMP activity. These results suggest that low oxygen tension increases endothelial cell capacity to produce NO in response to EPO by induction of both EPOR and eNOS. This effect of EPO on eNOS may be a physiologically relevant mechanism to counterbalance the hypertensive effects of increased hemoglobin-related NO destruction resulting from hypoxia-induced increased red cell mass.

Topics: Cardiovascular Diseases; Cell Differentiation; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Erythropoietin; Hemoglobins; Humans; Hypoxia; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitrites; Oligonucleotides; Oxygen; Receptors, Erythropoietin; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; Time Factors; Ultraviolet Rays

We hypothesized that the phosphodiesterase 5 inhibitor, sildenafil, and the guanosine cyclase stimulator, atrial natriuretic peptide (ANP), would act synergistically to increase cGMP levels and blunt hypoxic pulmonary hypertension in rats, because these compounds act via different mechanisms to increase the intracellular second messenger. Acute hypoxia: Adult Sprague-Dawley rats were gavaged with sildenafil (1 mg/ kg) or vehicle and exposed to acute hypoxia with and without ANP (10(-8)-10(-5) M ). Sildenafil decreased systemic blood pressure (103 +/- 10 vs. 87 +/- 6 mm Hg, P < 0.001) and blunted the hypoxia-induced increase in right ventricular systolic pressure (RVSP; percent increase 73.7% +/- 9.4% in sildenafil-treated rats vs. 117.2% +/- 21.1% in vehicle-treated rats, P = 0.03). Also, ANP and sildenafil had synergistic effects on blunting the hypoxia-induced increase in RVSP (P < 0.001) and on rising plasma cGMP levels (P < 0.05). Chronic hypoxia: Other rats were exposed to prolonged hypoxia (3 weeks, 0.5 atm) after subcutaneous implantation of a sustained-release pellet containing lower (2.5 mg), or higher (25 mg) doses of sildenafil, or placebo. Higher-dose, but not lower-dose sildenafil blunted the chronic hypoxia-induced increase in RVSP (P = 0.006). RVSP and plasma sildenafil levels were inversely correlated in hypoxic rats (r(2) = 0.68, P = 0.044). Lung cGMP levels were increased by both chronic hypoxia and sildenafil, with the greatest increase achieved by the combination. Plasma and right ventricular (RV) cGMP levels were increased by hypoxia, but sildenafil had no effect. RV hypertrophy and pulmonary artery muscularization were also unaffected by sildenafil. In conclusion, sildenafil and ANP have synergistic effects on the blunting of hypoxia-induced pulmonary vasoconstriction. During chronic hypoxia, sildenafil normalizes RVSP, but in the doses used, sildenafil has no effect on RV hypertrophy or pulmonary vascular remodeling.

Topics: Acute Disease; Animals; Atrial Natriuretic Factor; Cyclic GMP; Drug Synergism; Humans; Hypertension, Pulmonary; Hypoxia; Phosphodiesterase Inhibitors; Piperazines; Purines; Rats; Rats, Sprague-Dawley; Sildenafil Citrate; Sulfones

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

1 When nitric oxide synthase (NOS) produces NO from N(G)-hydroxy-L-arginine (OH-arginine) instead of L-arginine, the total requirement of molecular oxygen and NADPH to form NO is reduced. The aim of this work was to evaluate the effects of OH-arginine on the contractility of rabbit corpus cavernosum (RCC) and to compare the capacities of L-arginine and OH-arginine to enhance NO-mediated responses under normoxic and hypoxic conditions and in ageing, as models of defective NO production. 2 OH-arginine, but not L-arginine, was able to relax phenylephrine-contracted rabbit trabecular smooth muscle. OH-arginine-induced relaxation was inhibited by the NOS-inhibitor, L-NNA (300 microM), and by the guanylyl cyclase inhibitor, ODQ (20 microM), while it was not affected by the cytochrome P450 oxygenase inhibitor, miconazole (0.1 mM). Administration of OH-arginine, but not L-arginine, produced a significant increment of cGMP accumulation in RCC tissue. 3 Relaxation elicited by OH-arginine (300 microM) was still observed at low oxygen tension. The increase of cGMP levels induced by ACh (30 microM) in RCC was significantly enhanced by addition of OH-arginine (300 microM) in normoxic conditions, as well as under hypoxia, while L-arginine did not alter the effects of ACh on cGMP accumulation. 4 Endothelium-dependent and nitrergic nerve-mediated relaxations were both significantly reduced in RCC from aged animals (>20-months-old) when compared with young adult rabbits (5-months-old). Treatment with OH-arginine (300 microM) significantly potentiated endothelium-dependent and neurogenic relaxation in corpus cavernosum from aged rabbits, while L-arginine (300 microM) did not have significant effects. 5 Results show that OH-arginine promotes NO-mediated relaxation of RCC and potentiates the NO-mediated responses induced by stimulation of endogenous NO generation in hypoxic and aged tissues. We propose that the use of OH-arginine could be of interest in the treatment of erectile dysfunction, at least in those secondary to defective NO production.

Topics: Aging; Animals; Arginine; Cyclic GMP; Dose-Response Relationship, Drug; Hydroxyl Radical; Hypoxia; In Vitro Techniques; Male; Nitric Oxide; Penis; Rabbits; Vasodilation

Hypoxia-evoked vasodilatation is a fundamental regulatory mechanism that is often attributed to adenosine. The identity of the O(2) sensor is unknown. Nitric oxide (NO) inhibits endothelial mitochondrial respiration and ATP generation by competing with O(2) for its binding site on cytochrome oxidase. We proposed that in vivo this interaction allows endothelial cells to release adenosine when O(2) tension falls or NO concentration increases. Using anaesthetised rats, we confirmed that the increase in femoral vascular conductance (FVC, hindlimb vasodilatation) evoked by systemic hypoxia is attenuated by NO synthesis blockade with L-NAME, but restored when baseline FVC is restored by infusion of NO donor. This "restored" hypoxic response, like the control hypoxic response, is inhibited by the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Similarly, the FVC increase evoked by adenosine infusion was attenuated by L-NAME but restored by infusion of NO donor. However, when baseline FVC was restored after L-NAME with 8-bromo-cGMP, the FVC increase evoked by adenosine infusion was restored, but not in response to systemic hypoxia, suggesting that adenosine was no longer released by hypoxia. Infusion of NO donor at a given rate after treatment with L-NAME evoked a greater FVC increase during systemic hypoxia than during normoxia, both responses being reduced by 8-cyclopentyl-1,3-dipropylxanthine. Finally, both bradykinin and NO donor released adenosine from superfused endothelial cells in vitro; L-NAME attenuated only the former response. We propose that in vivo, shear-released NO increases the apparent K(m) of endothelial cytochrome oxidase for O(2), allowing the endothelium to act as an O(2) sensor, releasing adenosine in response to moderate falls in O(2).

Topics: Adenosine; Animals; Cyclic GMP; Endothelium, Vascular; Enzyme Inhibitors; Hindlimb; Hypoxia; In Vitro Techniques; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Purinergic P1 Receptor Antagonists; Rats; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine; Vasodilation; Vasodilator Agents; Xanthines

Phosphodiesterase type 5 (PDE5) inhibitors (eg, sildenafil) are a novel, orally active approach to the treatment of pulmonary arterial hypertension. The role of natriuretic peptides in the response to sildenafil was examined in mice lacking NPR-A, a guanylyl cyclase-linked natriuretic peptide receptor, in which pulmonary hypertension was induced by hypoxia.. Mice homozygous for NPR-A (NPR-A+/+) and null mutants (NPR-A-/-) were studied. Sildenafil inhibited the pressor response to acute hypoxia in the isolated perfused lungs of both genotypes. This effect was greater in the presence of atrial natriuretic peptide in the perfusate in NPR-A+/+ mice but not NPR-A-/- animals. In vivo, NPR-A mutants had higher basal right ventricular (RV) systolic pressures (RVSPs) than did NPR-A+/+ mice, and this was not affected by 3 weeks of treatment with sildenafil (25 mg x kg(-1) x d(-1)). Both genotypes exhibited a rise in RVSP and RV weight with chronic hypoxia (10% O2 for 21 days); RVSP and RV weight were reduced by continuous sildenafil administration in NPR-A+/+ mice, but only RVSP showed evidence of a response to the drug in NPR-A-/- mice. The effect of sildenafil on hypoxia-induced pulmonary vascular muscularization and cyclic GMP levels was also blunted in NPR-A-/- mice.. The natriuretic peptide pathway influences the response to PDE5 inhibition in hypoxia-induced pulmonary hypertension, particularly its effects on RV hypertrophy and vascular remodeling.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Atrial Natriuretic Factor; Blood Pressure; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Disease Models, Animal; Guanylate Cyclase; Homozygote; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; In Vitro Techniques; Lung; Mice; Mice, Mutant Strains; Perfusion; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Piperazines; Purines; Receptors, Atrial Natriuretic Factor; Respiration, Artificial; Sildenafil Citrate; Sulfones; Ventricular Function, Right

Recent studies from our laboratory indicate that pulmonary vasodilatory responses to exogenous nitric oxide (NO) are attenuated following chronic hypoxia (CH) and that this NO-dependent vasodilation is mediated by cGMP. Similarly, we have demonstrated that CH attenuates vasodilatory responses to the cGMP analog 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). We hypothesized that attenuated pulmonary vasodilation to 8-BrcGMP following CH is mediated by decreased protein kinase G-1 (PKG-1) expression/activity. Therefore, we examined vasodilatory responses to 8-BrcGMP (1 microM) in isolated, saline-perfused lungs from control and CH (4 wk at barometric pressure of 380 mmHg) rats in the presence of the competitive PKG inhibitor Rp-beta-phenyl-1, N2-etheno-8-bromoguanosine 3',5'-cyclic monophosphorothionate (30 microM) or the highly specific PKG inhibitor KT-5823 (10 microM). PKG-1 expression and activity were determined in whole lung homogenates from each group, and vascular PKG-1 levels were assessed by quantitative immunohistochemistry. PKG inhibition with either Rp-8-Br-PET-cGMPS or KT-5823 diminished vasodilatory responses to 8-BrcGMP in lungs from both control and CH rats, thus indicating a role for PKG in mediating reactivity to 8-BrcGMP in each group. However, in contrast to our hypothesis, PKG-1 levels were approximately twofold greater in lungs from CH rats vs. controls, and furthermore, this upregulation was localized to the vasculature. This correlates with an increase in PKG activity following CH. We conclude that PKG-1 is involved in 8-BrcGMP-mediated vasodilation; however, attenuated pulmonary vasodilation following CH is not associated with decreased expression/activity of PKG-1.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Alkaloids; Animals; Carbazoles; Chronic Disease; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Immunohistochemistry; Indoles; Lung; Nitric Oxide; Platelet Aggregation Inhibitors; Polycythemia; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Thionucleotides; Up-Regulation; Vascular Resistance; Vasodilation

In order to compare the effects of inhaled nitric oxide (NO) and magnesium (Mg) and their potential synergism on hemodynamics in pulmonary hypertension, we compared the effect of NO and Mg alone and in combination. The fact that simultaneous administration selectively increases pulmonary vascular relaxation would suggest that their association would allow for a decrease in the NO concentration required for optimal pulmonary vasodilation, thus lowering the risk of side effects.. We compared the effects of 20 ppm inhaled NO, 25 mg/kg Mg, and combined therapy in a pig model of hypoxic pulmonary hypertension. Twelve animals were randomly assigned to one of 3 treatment groups: control (group 1); Mg+NO group, receiving Mg followed by NO 15 min later (group 2); NO+Mg group, receiving NO followed by Mg 15 min later (group 3). The cardiac index, pulmonary and systemic pressures, pulmonary and systemic resistance, and the pulmonary to systemic resistance ratio (PVR/SVR) were recorded at baseline, on hypoxia and 15 minutes after each drug alone and in combination.. PVR/SVR decreased with NO alone (0.32+/-0.07 to 0.18+/-0.04; p<0.05) but not with Mg alone. When NO was added to Mg, PVR/SVR decreased significantly (0.31+/-0.06 to 0.16+/-0.02; p<0.05) but not when Mg was added to NO.. Inhaled NO is a selective pulmonary vasodilator in a pig model of hypoxic pulmonary hypertension. The simultaneous administration of intravenous Mg does not enhance the selective pulmonary vasodilation induced by NO inhalation.

Topics: Administration, Inhalation; Animals; Cyclic GMP; Disease Models, Animal; Drug Therapy, Combination; Female; Heart Rate; Hypertension, Pulmonary; Hypoxia; Injections, Intravenous; Magnesium; Magnesium Sulfate; Nitric Oxide; Oxygen; Partial Pressure; Swine; Vascular Resistance

To investigate the role of endogenous carbon monoxide (CO) in hypoxia.. After rats were inhaled with hypoxic gases and the heme oxygenase inhibitor ZnPPIX was administered, we measured the CO levels in plasma, liver, lung and kidney. Meanwhile plasma cGMP levels were observed. Furthermore, we recorded the change of hemodynamic and blood gases.. Acute mild hypoxia (10% O2) significantly increaed CO levels in plasma as well as liver, kidney and lung, while acute severe hypoxia (5% O2) significantly decreased CO levels in plasma as well as liver, kidney and lung. In addition, the former significantly elevated cGMP levels in plasma while the latter markedly reduced cGMP levels in plasma. The hemodynamic change occurred in accordance with the changes carbon monoxide.. Our results indicate, for the first time, that the endogenous carbon monoxide plays an important role in regulating the vessel tone during hypoxia.

Topics: Acute Disease; Animals; Blood Gas Analysis; Carbon Monoxide; Cyclic GMP; Heme Oxygenase (Decyclizing); Hemodynamics; Hypoxia; Kidney; Liver; Lung; Rats; Rats, Wistar

Hypoxic pulmonary vasoconstriction (HPV) is encountered during ascent to high altitude. Atrial natriuretic peptide (ANP) could be an option to treat HPV because of its natriuretic, diuretic, and vasodilatory properties. Data on effects of ANP on pulmonary and systemic circulation during HVP are conflicting, partly owing to anesthesia, surgical stress or uncontrolled dietary conditions. Therefore, ten conscious, chronically tracheotomized dogs were studied under standardized dietary conditions. The dogs were trained to breathe spontaneously at a ventilator circuit.. 30min of normoxia [inspiratory oxygen fraction (F(i)O(2))=0.21] were followed by 30min of hypoxia without ANP infusion (Hypoxia I, F(i)O(2)=0.1). While maintaining hypoxia an intravenous infusion of atrial natriuretic peptide was started with 50ng x kg body wt(-1) x min(-1) for 30min (Hypoxia+ANP1=low dose), followed by 1000ng x kg body wt(-1) x min(-1) for 30min (Hypoxia+ANP2=high dose). Thereafter, ANP infusion was stopped and hypoxia maintained for a final 30min (Hypoxia II). Compared to normoxia, mean pulmonary arterial pressure (MPAP) (16+/-0.7 vs. 26+/-1.3mmHg) and pulmonary vascular resistance (PVR) (448+/-28 vs. 764+/-89dyn x s(-1) x cm(-5)) increased during Hypoxia I and decreased during Hypoxia+ANP 1 (MPAP 20+/-1mmHg, PVR 542+/-55dyn x s(-1) x cm(-5)) (P<0.05). The higher dose of ANP did not further decrease MPAP or PVR, but started to have a tendency to decrease mean arterial pressure and cardiac output. We conclude that low dose ANP is able to reduce HPV without affecting systemic circulation during acute hypoxia.

Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Blood Circulation; Blood Gas Analysis; Cyclic GMP; Dogs; Drug Administration Schedule; Hemodynamics; Hydrogen-Ion Concentration; Hypoxia; Infusions, Intravenous; Oxygen; Pulmonary Artery; Pulmonary Circulation; Pulmonary Gas Exchange; Renin; Time Factors; Vascular Resistance; Vasoconstriction

To characterize the effects of ischemia on cGMP synthesis in microvascular endothelium, cultured endothelial cells from adult rat hearts were exposed to hypoxia or normoxia at pH 6.4 or 7.4. Cellular cGMP and soluble (sGC) and membrane guanylyl cyclase (mGC) activities were measured after stimulation of sGC (S-nitroso-N-acetyl-penicillamine) or mGC (urodilatin) or after no stimulation. Cell death (lactate dehydrogenase release) was negligible in all experiments. Hypoxia at pH 6.4 induced a rapid approximately 90% decrease in cellular cGMP after sGC and mGC stimulation. This effect was reproduced by acidosis. Hypoxia at pH 7.4 elicited a less pronounced (approximately 50%) and slower reduction in cGMP synthesis. Reoxygenation after 2 h of hypoxia at either pH 6.4 or 7.4 normalized the response to mGC stimulation but further deteriorated the sGC response; normalization of pH rapidly reversed the effects of acidosis. At pH 7.4, the response to GC stimulation correlated well with cellular ATP. We conclude that simulated ischemia severely depresses cGMP synthesis in microvascular coronary endothelial cells through ATP depletion and acidosis without intrinsic protein alteration.

Topics: Acidosis; Adenosine Triphosphate; Animals; Atrial Natriuretic Factor; Coronary Vessels; Cyclic GMP; Endothelium, Vascular; Energy Metabolism; Enzyme Activation; Guanylate Cyclase; Hydrogen-Ion Concentration; Hypoxia; Male; Microcirculation; Nitric Oxide; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reperfusion Injury

In the pulmonary artery isolated from 1-week hypoxia-induced pulmonary hypertensive rats, endothelial NO production stimulated by carbachol was decreased significantly in in situ visualization using diaminofluorescein-2 diacetate and also in cGMP content. This change was followed by the decrease in carbachol-induced endothelium-dependent relaxation. Protein expression of endothelial NO synthase (eNOS) and its regulatory proteins, caveolin-1 and heat shock protein 90, did not change in the hypoxic pulmonary artery, indicating that chronic hypoxia impairs eNOS activity at posttranslational level. In the hypoxic pulmonary artery, the increase in intracellular Ca(2+) level stimulated by carbachol but not by ionomycin was reduced. We next focused on changes in Ca(2+) sensitivity of the eNOS activation system. A morphological study revealed atrophy of endothelial cells and a peripheral condensation of eNOS in hypoxic endothelial cells preserving co-localization between eNOS and Golgi or plasma membranes. However, eNOS was tightly coupled with caveolin-1, and was dissociated from heat shock protein 90 or calmodulin in the hypoxic pulmonary artery in either the presence or absence of carbachol. Furthermore, eNOS Ser(1177) phosphorylation in both conditions significantly decreased without affecting Akt phosphorylation in the hypoxic artery. In conclusion, chronic hypoxia impairs endothelial Ca(2+) metabolism and normal coupling between eNOS and caveolin-1 resulted in eNOS inactivity.

Topics: Animals; Blotting, Western; Body Weight; Calcium; Carbachol; Cell Membrane; Cyclic GMP; Electrophoresis, Polyacrylamide Gel; Endothelium, Vascular; Golgi Apparatus; HSP90 Heat-Shock Proteins; Hypertension, Pulmonary; Hypoxia; Microscopy, Fluorescence; Muscle Contraction; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Organ Size; Phosphorylation; Precipitin Tests; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Serine; Time Factors

Hypoxia evokes a regulated decrease in body temperature, a response that has been termed anapyrexia, but the mechanisms involved are poorly understood. Therefore, the present study was undertaken to test the hypothesis that hypoxia-induced anapyrexia results from the activation of cAMP- and cGMP-dependent pathways in the preoptic region (PO). Adult male Wistar rats weighing 230-260 g were used. Body temperature was monitored by biotelemetry, and the levels of cAMP and cGMP were determined in the anteroventral third ventricular region (AV3V), where the PO is located. Using immunohistochemistry, we observed that the PO contains a high density of cAMP- and cGMP-containing cells. Interestingly, hypoxia exposure raised the levels of cAMP and cGMP in the AV3V. Intra-PO microinjection of Rp-cAMPS, an inhibitor of cAMP-dependent protein kinase, attenuated hypoxia-induced anapyrexia. Similarly, intra-PO microinjection of the mixed beta-adrenoceptor/serotonin (5-HT(1A)) receptor antagonist propranolol also impaired the drop in body temperature in response to hypoxia. The reduction in body temperature evoked by intra-PO serotonin, but not epinephrine, was blocked by Rp-cAMPS, indicating the involvement of a preoptic serotonin-cAMP pathway in the development of anapyrexia. Moreover, microinjection of N(G)-monomethyl-l-arginine, an inhibitor of nitric oxide (NO) synthesis, or Rp-cGMPS, an inhibitor of cGMP-dependent protein kinase, into the PO also attenuated hypoxia-induced anapyrexia. In conclusion, the present study supports that hypoxia-induced anapyrexia results from the activation of the serotonin-cAMP and NO-cGMP pathways in the PO.

Topics: Animals; Body Temperature Regulation; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Epinephrine; Hypoxia; Male; Microinjections; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Preoptic Area; Propranolol; Rats; Rats, Wistar; Serotonin; Signal Transduction; Time Factors; Vasodilation

The change in the content of cyclic GMP, cyclic AMP, ATP, ADP, AMP and fructose-2,6-bisphosphate that occurred in the mantle of the mussel Mytilus galloprovincialis Lmk when specimens of this mollusk were subjected to a hypoxia/anoxia situation were assessed. After the early 24 h in anaerobiosis, a clear decrease was observed in the ATP content, which remained close to that value for the rest of the time. AMP content doubled during the early 24 h in anaerobiosis and, from that time on, it remained close to that value. Fructose-2,6-bisphoshate and cyclic GMP showed a similar behavior. The levels of these compounds rose significantly during the early hours in anaerobiosis, and then fell to values similar to those of aerobiosis, remaining constant for the rest of the time. Neither ADP nor cAMP showed significant variations.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Bivalvia; Cyclic AMP; Cyclic GMP; Fructosediphosphates; Glycolysis; Hypoxia; Phosphates

To determine the effects of simulated intermittent high altitude hypoxia adaptation (IHA) on coronary capillary and coronary flow (CF) in rat hearts.. Model of Langendorf-perfused isolated rat hearts were used to measure CF during ischemia-reperfusion, and immunoperoxidase staining assay and computer-aid morphometry analysis were conducted to determine the myocardial capillary densities. Cyclic GMP (cGMP) level in myocardium was measured by radio-immunoassay.. Pre-ischemia level of CF in IHA rats was higher (IHA28 13.4 mL/min+/-1.5 mL/min, IHA42 15.4 mL/min+/-2.0 mL/min, P < 0.01) than that of normoxic rats (11.0+/-0.8) mL/min, and the recovery of CF after ischemia-reperfusion was better in IHA rats. As an adaptive result, the myocardial capillary densities of the left ventricular myocardium in IHA rats were 1.5 times of those in normoxic control rats, but there was no apparent ventricular hypertrophy in IHA rats. Myocardial cGMP content (1.8+/-0.7) nmol/g in IHA rats were increased significantly compared with control rats (1.1+/-0.4) nmol/g, but cGMP level was not altered before and after ischemia-reperfusion in either group. It was also revealed that in isolated rat hearts perfused, myocardial function recovered better in IHA rats than that in normoxic control rats.. IHA adaptation increased the tolerance of rat hearts against subsequent ischemia-reperfusion injury, and increase in coronary circulation and angiogenesis might be the mechanisms of myocardium protected by IHA.

Topics: Adaptation, Physiological; Altitude Sickness; Animals; Coronary Circulation; Cyclic GMP; Hypoxia; In Vitro Techniques; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Neovascularization, Physiologic; Random Allocation; Rats; Rats, Sprague-Dawley; Regional Blood Flow

Heparin and nitric oxide (NO) attenuate changes to the pulmonary vasculature caused by prolonged hypoxia. Heparin may increase NO; therefore, we hypothesized that heparin may attenuate hypoxia-induced pulmonary vascular remodeling via a NO-mediated mechanism. In vivo, rats were exposed to normoxia (N) or hypoxia (H; 10% O(2)) with or without heparin (1,200 U x kg-1 x day-1) and/or the NO synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME; 20 mg x kg-1 x day-1) for 3 days or 3 wk. Heparin attenuated increases in pulmonary arterial pressure, the percentage of muscular pulmonary vessels, and their medial thickness induced by 3 wk of H. Importantly, although L-NAME alone had no effect, it prevented these effects of heparin on vascular remodeling. In H lungs, heparin increased NOS activity and cGMP levels at 3 days and 3 wk and endothelial NOS protein expression at 3 days but not at 3 wk. In vitro, heparin (10 and 100 U x kg-1 x ml-1) increased cGMP levels after 10 min and 24 h in N and anoxic (0% O2) endothelial cell-smooth muscle cell (SMC) coculture. SMC proliferation, assessed by 5-bromo-2'-deoxyuridine incorporation during a 3-h incubation period, was decreased by heparin under N, but not anoxic, conditions. The antiproliferative effects of heparin were not altered by L-NAME. In conclusion, the in vivo results suggest that attenuation of hypoxia-induced pulmonary vascular remodeling by heparin is NO mediated. Heparin increases cGMP in vitro; however, the heparin-induced decrease in SMC proliferation in the coculture model appears to be NO independent.

Topics: Animals; Cell Division; Cells, Cultured; Coculture Techniques; Cyclic GMP; Endothelium, Vascular; Enzyme Activation; Enzyme Inhibitors; Heart Ventricles; Heparin; Hypoxia; Lung; Male; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Organ Size; Pulmonary Circulation; Pulmonary Wedge Pressure; Rats; Rats, Sprague-Dawley; Time

Chronic hypoxia (CH) augments endothelium-derived nitric oxide (NO)-dependent pulmonary vasodilation; however, responses to exogenous NO are reduced following CH in female rats. We hypothesized that CH-induced attenuation of NO-dependent pulmonary vasodilation is mediated by downregulation of vascular smooth muscle (VSM) soluble guanylyl cyclase (sGC) expression and/or activity, increased cGMP degradation by phosphodiesterase type 5 (PDE5), or decreased VSM sensitivity to cGMP. Experiments demonstrated attenuated vasodilatory responsiveness to the NO donors S-nitroso-N-acetylpenicillamine and spermine NONOate and to arterial boluses of dissolved NO solutions in isolated, saline-perfused lungs from CH vs. normoxic female rats. In additional experiments, the sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, blocked vasodilation to NO donors in lungs from each group. However, CH was not associated with decreased pulmonary sGC expression or activity as assessed by Western blotting and cGMP radioimmunoassay, respectively. Consistent with our hypothesis, the selective PDE5 inhibitors dipyridamole and T-1032 augmented NO-dependent reactivity in lungs from CH rats, while having little effect in lungs from normoxic rats. However, the attenuated vasodilatory response to NO in CH lungs persisted after PDE5 inhibition. Furthermore, CH similarly inhibited vasodilatory responses to 8-bromoguanosine 3'5'-cyclic monophosphate. We conclude that attenuated NO-dependent pulmonary vasodilation after CH is not likely mediated by decreased sGC expression, but rather by increased cGMP degradation by PDE5 and decreased pulmonary VSM reactivity to cGMP.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Blotting, Western; Chronic Disease; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 5; Enzyme Inhibitors; Female; Guanylate Cyclase; Hypertrophy, Right Ventricular; Hypoxia; In Vitro Techniques; Lung; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Donors; Nitroarginine; Polycythemia; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Vasodilation

We tested the hypothesis that chronic high-altitude (3,820 m) hypoxia during pregnancy was associated with the upregulation of endothelial nitric oxide (NO) synthase (eNOS) protein and mRNA in ovine uterine artery endothelium and enhanced endothelium-dependent relaxation. In pregnant sheep, norepinephrine-induced dose-dependent contractions were increased by removal of the endothelium in both control and hypoxic uterine arteries. The increment was significantly higher in hypoxic tissues. The calcium ionophore A23187-induced relaxation of the uterine artery was significantly enhanced in hypoxic compared with control tissues. However, sodium nitroprusside- and 8-bromoguanosine 3',5'-cyclic monophosphate-induced relaxations were not changed. Accordingly, chronic hypoxia significantly increased basal and A23187-induced NO release. Chronic hypoxia increased eNOS protein and mRNA levels in the endothelium from uterine but not femoral or renal arteries. In nonpregnant animals, chronic hypoxia increased eNOS mRNA in uterine artery endothelium but had no effects on eNOS protein, NO release, or endothelium-dependent relaxation. Chronic hypoxia selectively augments pregnancy-associated upregulation of eNOS gene expression and endothelium-dependent relaxation of the uterine artery.

Topics: Altitude Sickness; Animals; Calcimycin; Chronic Disease; Cyclic GMP; Endothelium, Vascular; Female; Femoral Artery; Gene Expression Regulation, Enzymologic; Hypoxia; Ionophores; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroprusside; Norepinephrine; Pregnancy; Pregnancy, Animal; Renal Artery; RNA, Messenger; Sheep; Umbilical Arteries; Uterus; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

Chronic myocardial hypoxia results in elevated nitric oxide (NO) production and increased current through the sarcolemmal K(ATP) channel. We hypothesized these two processes are related and determined whether NO alters the electrophysiology of Purkinje fibers obtained from rabbits (n=12/group) raised in a normoxic (F(I)O2=0.21) or hypoxic (F(I)O2=0.12) environment from birth to 9 days of age. Action potential duration (APD)(90) was shorter (112+/-3 ms v 126+/-3 ms) and maximum diastolic potential (MDP) was more negative (-84+/-2 mV v-80+/-1 mV) in hypoxic hearts compared with normoxic controls. In normoxic hearts the NO donors, S-nitrosoglutathione (GSNO) 50 microM and spermine NONOate (50 microM) shortened APD(90) and increased MDP to levels present in chronically hypoxic hearts. This effect was completely abolished by the K(ATP) channel blocker glibenclamide (3 microM) and by a nitric oxide trap, Carboxy-PTIO (100 microM). The NO carrier glutathione (50 microM) and decomposed spermine NONOate had no effect on APD(90) or MDP. GSNO had no effect in hypoxic hearts; however, when GSNO was combined with glibenclamide APD(90) increased, and MDP decreased to normoxic values. 8-Bromo cGMP (100 microM) shortened APD(90) and increased MDP to levels present in chronically hypoxic hearts. This effect was abolished by glibenclamide. A soluble guanylyl cyclase inhibitor, ODQ (10 microM), had no effect on action potentials in normoxic hearts but in hypoxic hearts resulted in an increase in APD(90) to levels present in normoxic hearts and a decrease in MDP. The effect of ODQ could not be reversed by GSNO. We conclude that NO activates the sarcolemmal K(ATP) channel in normoxic and chronically hypoxic hearts by a cyclic GMP-dependent mechanism.

Topics: Action Potentials; Animals; Animals, Newborn; Blood Pressure; Cyclic GMP; Electron Spin Resonance Spectroscopy; Electrophysiology; Female; Glutathione; Glyburide; Hypoglycemic Agents; Hypoxia; Male; Models, Biological; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Nitroso Compounds; Oxygen; Potassium Channels; Rabbits; S-Nitrosoglutathione; Sarcolemma; Spermine; Time Factors

We examined whether overproduction of endogenous nitric oxide (NO) can prevent hypoxia-induced pulmonary hypertension and vascular remodeling by using endothelial NO-overexpressing (eNOS-Tg) mice. Male eNOS-Tg mice and their littermates (wild-type, WT) were maintained in normoxic or 10% hypoxic condition for 3 weeks. In normoxia, eNOS protein levels, Ca(2+)-dependent NOS activity, and cGMP levels in the lung of eNOS-Tg mice were higher than those of WT mice. Activity of eNOS and cGMP production in the lung did not change significantly by hypoxic exposure in either genotype. Chronic hypoxia did not induce iNOS expression nor increase its activity in either genotype. Plasma and lung endothelin-1 levels were increased by chronic hypoxia, but these levels were not significantly different between the 2 genotypes. In hemodynamic analysis, right ventricular systolic pressure (RVSP) in eNOS-Tg mice was similar to that in WT mice in normoxia. Chronic hypoxia increased RVSP and induced right ventricular hypertrophy in both genotypes; however, the degrees of these increases were significantly smaller in eNOS-Tg mice. Histological examination revealed that hypoxic mice showed medial wall thickening in pulmonary arteries. However, the increase of the wall thickening in small arteries (diameter <80 microm) by chronic hypoxia was inhibited in eNOS-Tg mice. Furthermore, muscularization of small arterioles was significantly attenuated in eNOS-Tg mice. Thus, we demonstrated directly that overproduction of eNOS-derived NO can inhibit not only the increase in RVSP associated with pulmonary hypertension but also remodeling of the pulmonary vasculature and right ventricular hypertrophy induced by chronic hypoxia.

Topics: Animals; Blood Pressure; Blood Vessels; Cyclic GMP; Endothelin-1; Female; Genotype; Heart Rate; Hematocrit; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Immunoblotting; Lung; Male; Mice; Mice, Transgenic; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Ventricular Function, Right; Ventricular Pressure

Nitric oxide (NO) is a potent vasodilator and inhibitor of vascular remodeling. Reduced NO production has been implicated in the pathophysiology of pulmonary hypertension, with endothelial NO synthase (NOS) knockout mice showing an increased risk for pulmonary hypertension. Because molecular oxygen (O2) is an essential substrate for NO synthesis by the NOSs and biochemical studies using purified NOS isoforms have estimated the Michaelis-Menten constant values for O2 to be in the physiological range, it has been suggested that O2 substrate limitation may limit NO production in various pathophysiological conditions including hypoxia. This review summarizes numerous studies of the effects of acute and chronic hypoxia on NO production in the lungs of humans and animals as well as in cultured vascular cells. In addition, the effects of hypoxia on NOS expression and posttranslational regulation of NOS activity by other proteins are also discussed. Most studies found that hypoxia limits NO synthesis even when NOS expression is increased.

Topics: Acute Disease; Animals; Chronic Disease; Cyclic GMP; Humans; Hypoxia; Lung; Nitric Oxide; Nitric Oxide Synthase; Oxygen

Endogenous as well as exogenous atrial natriuretic peptide (ANP) attenuates the development of chronic hypoxic pulmonary hypertension (CHPH) in rats. We built a recombinant adenovirus type 5 containing ANP cDNA under the control of the Rous sarcoma virus long terminal repeat (Ad.ANP). The efficiency of this vector in delivering the ANP gene was first examined in rat primary cultures of pulmonary vessel smooth muscle cells (SMCs) in comparison with Ad.beta GAL. Conditioned medium collected from Ad.ANP-infected cells (1000 TCID(50)/cell) contained 5 x 10(9) M immunoreactive ANP and elicited relaxation of isolated rat pulmonary arteries preconstricted with phenylepinephrine. To examine the effects of adenovirus-mediated ANP expression in the CHPH rat lung, Ad.ANP or Ad.beta GAL was administered via the tracheal route. Immunoreactive ANP was detected in bronchoalveolar fluid as early as 4 days and until 10-17 days after Ad.ANP administration (5 x 10(8) TCID(50)). Lung ANP immunostaining was mainly localized in bronchial and alveolar epithelial cells. As compared with Ad.beta GAL-treated controls, rats given Ad.ANP (5 x 10(8) TCID(50)) on the day before a 2-week exposure to hypoxia (10% O(2)) had lower values for pulmonary artery pressure (32.1 +/- 1.93 vs. 35.5 +/- 2 mmHg, p < 0.01) and Fulton's index (0.52 +/- 0.089 vs. 0.67 +/- 0.12, p < 0.001) and less severe right ventricular hypertrophy and distal vessel muscularization. These results suggest that induction of ANP expression in the lung may hold promise in the treatment of pulmonary hypertension.

Topics: Adenoviridae; Animals; Atrial Natriuretic Factor; Avian Sarcoma Viruses; Body Weight; Bronchoalveolar Lavage Fluid; Cells, Cultured; Culture Media, Conditioned; Cyclic GMP; DNA, Complementary; Dose-Response Relationship, Drug; Epinephrine; Gene Transfer Techniques; Hypertension, Pulmonary; Hypoxia; Immunohistochemistry; Lung; Muscle, Smooth; Rats; Rats, Wistar; RNA, Messenger; Time Factors; Tissue Distribution; Trachea; Transfection; Transgenes

We have previously demonstrated that expression of the atrial natriuretic peptide (ANP) clearance receptor (NPR-C) is reduced selectively in the lung of rats and mice exposed to hypoxia but not in pulmonary arterial smooth muscle cells (PASMCs) cultured under hypoxic conditions. The current study tested the hypothesis that hypoxia-responsive growth factors, fibroblast growth factors (FGF-1 and FGF-2) and platelet-derived growth factor-BB (PDGF-BB), that activate tyrosine kinase receptors can reduce expression of NPR-C in PASMCs independent of environmental oxygen tension. Growth-arrested rat PASMCs were incubated under hypoxic conditions (1% O2) for 24 h; with FGF-1, FGF-2, or PDGF-BB (0.1-20 ng/ml for 1-24 h); or with ANG II (1-100 nM), endothelin-1 (ET-1, 0.1 microM), ANP (0.1 microM), sodium nitroprusside (SNP, 0.1 microM), or 8-bromo-cGMP (0.1 mM) for 24 h under normoxic conditions. Steady-state NPR-C mRNA levels were assessed by Northern blot analysis. FGF-1, FGF-2, and PDGF-BB induced dose- and time-dependent reduction of NPR-C mRNA expression within 1 h at a threshold concentration of 1 ng/ml; hypoxia, ANG II, ET-1, ANP, SNP, or cGMP did not decrease NPR-C mRNA levels in PASMCs under the above conditions. Downregulation of NPR-C expression by FGF-1, FGF-2, and PDGF-BB was inhibited by the selective FGF-1 receptor tyrosine kinase inhibitor PD-166866 and mitogen-activated protein/extracellular signal-regulated kinase inhibitors U-0126 and PD-98059. These results indicate that activation of tyrosine kinase receptors by hypoxia-responsive growth factors, but neither hypoxia per se nor activation of G protein-coupled receptors, inhibits NPR-C gene expression in PASMCs. These results suggest that FGF-1, FGF-2, and PDGF-BB play a role in the signal transduction pathway linking hypoxia to altered NPR-C expression in lung.

Topics: Animals; Cattle; Cell Division; Cells, Cultured; Cyclic GMP; Down-Regulation; Enzyme Inhibitors; Fetal Blood; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; GTP-Binding Proteins; Guanylate Cyclase; Hypoxia; Male; Mitogen-Activated Protein Kinase Kinases; Muscle, Smooth, Vascular; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Receptor Protein-Tyrosine Kinases; Receptors, Atrial Natriuretic Factor; Receptors, Fibroblast Growth Factor

In intrapulmonary arteries cultured under hypoxic conditions (5% oxygen) for 7 days, endothelium-dependent relaxation and cGMP accumulation induced by substance P were decreased as compared to those of a normoxic control (20% oxygen). In rabbit mesenteric arteries exposed to chronic hypoxia, however, endothelial dysfunction was not observed. Furthermore, in endothelium-denuded pulmonary arteries exposed to hypoxia, neither relaxation nor cGMP accumulation due to sodium nitroprusside differed from those of the normoxic control. Hypoxia did not change the mRNA expression of endothelial NO synthase (eNOS), the protein expression of eNOS or the eNOS regulatory protein caveolin-1 as assessed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) or whole-mount immunostaining. Morphological study revealed atrophy of endothelial cells and condensation of the eNOS protein in many cells. These results suggest that chronic hypoxia impaired NO-mediated arterial relaxation without changing either the eNOS protein expression or the NO-sensitivity of smooth muscle cells in pulmonary arteries. Changes in cell structure and organization may be involved in endothelial dysfunction.

Topics: Animals; Arginine; Biopterins; Caveolin 1; Caveolins; Cyclic GMP; Dinoprost; Dose-Response Relationship, Drug; Endothelium, Vascular; Hypoxia; Ionomycin; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroprusside; Organ Culture Techniques; Pulmonary Artery; Rabbits; RNA, Messenger; Substance P; Superoxide Dismutase; Vasoconstriction; Vasodilation; Vasodilator Agents

It was hypothesized that hypoxia may inhibit nitric oxide (NO) production by reducing the availability of endothelial NO synthase (NOS III) substrate. To evaluate the effect of L-arginine on the NO release in high altitude, 11 subjects were infused with L-arginine (0.5 g x kg(-1)) during 30 min in normoxia and after 36 h at 4,350 m (hypoxia). The L-citrulline and cyclic guanosine monophosphate (cGMP) concentrations were measured to investigate NO synthesis and guanylyl cyclase activity respectively. L-citrulline concentration, arterial oxygen saturation (Sa,O2), systemic blood pressure, heart rate and acute mountain sickness (AMS) score were measured at rest and 15, 30 and 45 min after starting infusion. The results showed that baseline L-citrulline was lower in hypoxia (p<0.05). L-arginine infusion increased L-citrulline concentration in both conditions. However, in hypoxia L-citrulline concentration remained lower than in normoxia (p<0.05). The concentration of cGMP was lower in hypoxia (p<0.05). In hypoxia, Sa,O2 increased from 15 min after the start of the infusion to 45 min (p<0.05). Blood pressure and heart rate were not affected by L-arginine infusion. Subjects who experienced symptoms of AMS showed a slight decrease in AMS score with L-arginine. The decreased L-citrulline suggests a hypoxia-induced impairment of nitric oxide synthase III or a decrease in L-arginine availability. The improvement of arterial oxygen saturation by pretreatment with L-arginine could be ascribed to an enhancement of the ventilation/perfusion ratio. Collectively, these results are consistent with a decrease in nitric oxide production in hypoxia that could be antagonized by supplying nitric oxide synthase cosubstrate.

Topics: Adult; Altitude; Altitude Sickness; Arginine; Citrulline; Cyclic GMP; Female; Humans; Hypoxia; Infusions, Intravenous; Linear Models; Male; Nitric Oxide; Oxygen; Radioimmunoassay; Reference Values; Time Factors

Differential screening of a hepatopancreas cDNA library derived from the marine snail Littorina littorea yielded a 421-bp clone coding for ribosomal protein L26 that was up-regulated during anoxia exposure. The deduced amino acid sequence, containing 144 residues with a predicted molecular weight of 17 kDa, showed 80% amino acid sequence identity to the mammalian ribosomal protein L26. Analysis of hepatopancreas and foot muscle samples from a time course of anoxia exposure showed a maximal transcript increase of 4- and 3-fold after 96 hr and 48 hr, respectively, relative to normoxic animals, with a subsequent decrease in transcript levels during normoxic recovery. Nuclear run-off assays confirmed the observed transcriptional up-regulation of L26 during anoxia. Organ culture experiments were performed to determine a possible pathway of up-regulation of L26, with data indicating a putative role for cGMP in signal transduction. The transcriptional up-regulation of L26 during anoxia may stabilize the existing mRNA pool, via a possible cGMP-mediated signaling cascade, until oxygen reappears and protein synthesis resumes.

Topics: Adaptation, Physiological; Animals; Cyclic GMP; Digestive System; Hypoxia; Muscles; Ribosomal Proteins; RNA, Messenger; Signal Transduction; Snails; Transcription, Genetic; Up-Regulation

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

Nitric oxide (NO) and dopamine (DA) have similar effects on renal function, with both having natriuretic and diuretic effects mediated by vascular and tubular mechanisms. Renal ischaemia or hypoxia have been shown to influence the activity of both systems. However, it is not known whether there is any crosstalk between the NO and dopaminergic systems in the kidney. Here using the porcine proximal tubule-like renal epithelial LLC-PK1 cell line as a model system, we determined whether exposure of cells to chemical hypoxia altered the steady-state levels of D1A receptor mRNA and whether the changes involved the NO system. Exposure of LLC-PK1 cells to chemical hypoxia resulted in a marked increase in D1A receptor mRNA levels as measured by reverse transcription-polymerase chain reaction (RT-PCR). The increased levels of D1A receptor mRNA following hypoxia were blocked by the NO synthase inhibitors NG-nitro-L-arginine methylester (L-NAME) or NG-monomethyl-L-arginine (L-NMMA). Further evidence that the NO system exerted positive effects on D1A receptor gene expression came from finding that the NO donor sodium nitroprusside, the NO precursor L-arginine and the guanosine 3', 5'-cyclic monophosphate (cyclic GMP) analogue 8-Br-cGMP all increased D1A receptor mRNA levels in LLC-PK1 cells. These results indicate that expression of the D1A receptor in LLC-PK1 cells can be positively regulated by the NO system. Such an interaction between the renal NO and DA systems may contribute to the reported protective effects that NO and DA exert upon the kidney under conditions of ischaemia.

Topics: Animals; Arginine; Cyclic GMP; Deoxyglucose; Enzyme Inhibitors; Epithelial Cells; Hypoxia; Kidney; LLC-PK1 Cells; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; omega-N-Methylarginine; Receptors, Dopamine D1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Cyanide; Swine

Calcitonin gene-related peptide (CGRP) is believed to play an important role in maintaining low pulmonary vascular resistance (PVR) and in modulating pulmonary vascular responses to chronic hypoxia; however, the effects of adenovirally mediated gene transfer of CGRP on the response to hypoxia are unknown.. In the present study, an adenoviral vector encoding prepro-CGRP (AdRSVCGRP) was used to examine the effects of in vivo gene transfer of CGRP on increases in PVR, right ventricular mass (RVM), and pulmonary vascular remodeling that occur in chronic hypoxia in the mouse. Intratracheal administration of AdRSVCGRP, followed by 16 days of chronic hypoxia (FIO(2) 0.10), increased lung CGRP and cAMP levels. The increase in pulmonary arterial pressure (PAP), PVR, RVM, and pulmonary vascular remodeling in response to chronic hypoxia was attenuated in animals overexpressing prepro-CGRP, whereas systemic pressure was not altered while in chronically hypoxic mice, angiotensin II and endothelin-1-induced increases in PAP were reduced, whereas decreases in PAP in response to CGRP and adrenomedullin were not changed and decreases in PAP in response to a cAMP phosphodiesterase inhibitor were enhanced by AdRSVCGRP.. In vivo CGRP lung gene transfer attenuates the increase in PVR and RVM, pulmonary vascular remodeling, and pressor responses in chronically hypoxic mice, suggesting that CGRP gene transfer alone and with a cAMP phosphodiesterase inhibitor may be useful for the treatment of pulmonary hypertensive disorders.

Topics: Adenoviridae; Adrenomedullin; Animals; beta-Galactosidase; Calcitonin Gene-Related Peptide; Cyclic AMP; Cyclic GMP; Endothelin-1; Genes, Reporter; Genetic Therapy; Genetic Vectors; Hemodynamics; Humans; Hypertension, Pulmonary; Hypoxia; Lung; Mice; NG-Nitroarginine Methyl Ester; Peptides; Phosphodiesterase Inhibitors; Potassium Channels; Protein Precursors; Purinones; Recombinant Fusion Proteins; Rolipram; Second Messenger Systems; Transfection; Vasoconstriction; Vasoconstrictor Agents; Vasodilator Agents

Using slices of the dorsal lateral geniculate nucleus, it has been shown that, in the presence of excitatory and inhibitory amino acid antagonists, brief periods of hypoxia (3-4 min of 95% N(2)/5% CO(2)) induce in thalamocortical neurons an increase in instantaneous input conductance (G(N)) accompanied by an inward shift in baseline holding current (I(BH)). These effects have been suggested to be mediated, at least in part, by a positive shift in the voltage-dependence of the hyperpolarization-activated, mixed Na(+)/K(+) current (I(h)) and a change in its activation kinetics which transforms it into an almost instantaneously activated current. In this study, using the whole-cell patch-clamp technique, the contribution of an increased Ca(2+)-dependent transmitter release to the hypoxic response of thalamocortical neurons was further investigated using (i) blockers of calcineurin, a Ca(2+)/calmodulin-activated phosphatase that selectively regulates Ca(2+)-dependent release, and (ii) antagonists of neurotransmitters that are known to modulate I(h). Thalamocortical neurons (n = 23) recorded with electrodes filled with calcineurin autoinhibitory fragment (30-250 microM), a membrane impermeable blocker of calcinuerin, showed no difference either in resting, or in the hypoxia-induced changes in, G(N), I(BH) and I(h), when compared to thalamocortical cells patched with electrodes that did not contain calcineurin autoinhibitory fragment. In contrast, in 18 of these neurons recorded with calcineurin autoinhibitory fragment-filled electrodes, bath application either of cyclosporin-A (20 microM) or tacrolimus (50-100 microM), two membrane permeable blockers of calcineurin, abolished the effects of hypoxia on G(N), I(BH), and I(h). Separate application of noradrenaline, serotonin, histamine and nitric oxide antagonists produced only a small depression of the hypoxic response, while concomitant bath application of these antagonists decreased the hypoxia-induced changes in G(N) and I(BH) by 55 and 42%, respectively (n = 12). Concomitant bath application of 8-bromo-adenosine-3'5'-cyclicmonophosphate and 8-bromo-guanosine-3'5'-cyclicmonophosphate (both 1mM), which are known to mediate the action of these transmitters on I(h), increased G(N) (40%), decreased I(h) time-constant of activation (30%) and significantly occluded (50%) the hypoxia-induced effect on G(N) and I(BH). Thalamocortical neurons (n = 6) patched with electrodes filled with 8-bromo-adenosine-3'5'-cyclicmon

Topics: Acute Disease; Animals; Calcineurin Inhibitors; Cyclic AMP; Cyclic GMP; Electrophysiology; Histamine Release; Hypoxia; Neurotransmitter Agents; Nitric Oxide; Norepinephrine; Rats; Rats, Wistar; Serotonin; Thalamic Diseases

The goal of this study was to determine whether hypoxia alters expression of endothelial nitric oxide synthase (eNOS) in the systemic circulation. Rats breathed either air or 10% oxygen for 12 hours, 48 hours, or 7 days. Thoracic aortas were excised and either mounted in organ bath myographs or frozen in liquid nitrogen for later extraction of protein and RNA. eNOS protein (Western blotting) was decreased (20% of normoxic control) after 12 hours, 48 hours, and 7 days of hypoxia. eNOS mRNA (ribonuclease protection assay) was similarly reduced. Acetylcholine (10(-4) mol/L) reversed phenylephrine (10(-5) mol/L) preconstriction by 53.3+/-5.6% in aortic rings from normoxic rats and 26.1+/-4.8% in rings from rats exposed to hypoxia for 48 hours (P<0.05), with comparable impairment of relaxation by the calcium ionophore A23187 (10(-5) mol/L). Responses to diethylamine nitric oxide and 8-bromo-cGMP were unaffected. Aortic cGMP levels after incubation with acetylcholine (10(-6) mol/L) averaged 14.0+/-1.8 fmol/mg in rings from normoxic rats compared with 8.7+/-1.0 fmol/mg in rings from hypoxic rats (P<0. 05). Similarly, nitrate concentration (by capillary electrophoresis) in the media in which the rings were incubated was reduced in the hypoxic group (5.6+/-0.23 micromol/L for hypoxic rats and 7.8+/-0.7 micromol/L for normoxic rats). Impaired endothelial NO release may handicap the vascular responses that defend vital organ function during hypoxia.

Topics: Animals; Aorta; Cyclic GMP; Down-Regulation; Endothelium, Vascular; Hypoxia; Male; Molecular Sequence Data; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Rats; Rats, Sprague-Dawley; RNA, Messenger; Time Factors; Vasodilation

Topics: Animals; Cattle; Cyclic GMP; Glutathione Peroxidase; Hydrogen Peroxide; Hypoxia; In Vitro Techniques; Models, Biological; Multienzyme Complexes; NADH, NADPH Oxidoreductases; Oxidation-Reduction; Oxygen; Pulmonary Artery; Signal Transduction; Vasodilation

We investigated the role of heme oxygenase (HO)-1 in the development of hypoxia-induced pulmonary hypertension. HO catalyzes the breakdown of heme to the antioxidant bilirubin and the vasodilator carbon monoxide. Hypoxia is a potent but transient inducer of HO-1 in vascular smooth muscle cells in vitro and in the lung in vivo. By using agonists of HO-1, we sustained a high expression of HO-1 in the lungs of rats for 1 week. We report that this in vivo enhancement of HO-1 in the lung prevented the development of hypoxic pulmonary hypertension and inhibited the structural remodeling of the pulmonary vessels. The mechanism(s) underlying this effect may involve a direct vasodilating and antiproliferative action of endogenous carbon monoxide, as well as an indirect effect of carbon monoxide on the production of vasoconstrictors. These results provide evidence that enhancement of endogenous adaptive responses may be used to prevent hypoxia-induced pulmonary hypertension.

Topics: Animals; Blood Circulation; Blood Vessels; Cyclic GMP; Gene Expression Regulation; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Hypertension, Pulmonary; Hypoxia; Lung; Male; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; RNA, Messenger

Activation of ATP sensitive K+ channels (K(ATP)) and the NO-cGMP pathway have both been implicated in reducing norepinephrine (NE) release from cardiac sympathetic nerves during stimulation. Our aim was to test whether these pathways could interact and modulate cardiac excitability during sympathetic nerve stimulation (SNS).. The effect of inhibitors and activators of K(ATP) channels and the NO-cGMP pathway on the heart rate (HR) response to cardiac SNS in the isolated guinea pig (Cavia porcellus) double atrial/right stellate ganglion preparation was studied (n=48).. The K(ATP) channel activator, diazoxide (100 microM, n=6) or hypoxia (0% O2/5% CO2, n=6) significantly attenuated the HR response to 3 Hz SNS by -10+/-4% and -27+/-6% respectively; an effect that was reversed by the K(ATP) channel inhibitor, glibenclamide (30 microM). Glibenclamide (n=6) on its own enhanced the HR response to SNS by 20+/-8%. Bath applied NE (0.1-0.7 microM, n=6) did not affect the HR response to diazoxide, although an increased response to glibenclamide was observed at 0.3 and 0.5 microM NE. In the presence of 8-Br-cGMP (0.5 mM, n=7), diazoxide further decreased the HR response SNS (19+/-3%). The NO synthase inhibitor, N-omega-nitro-L-arginine (100 microM) significantly increased the HR response (13+/-3%) to SNS in the presence of diazoxide (100 microM, n=6). This effect was reversed with excess (1 mM) L-arginine. Conversely, the NO donor, sodium nitroprusside (SNP, 20-100 microM) significantly attenuated the HR response to SNS. The addition of glibenclamide (30 microM, n=10) could still enhance the HR response (42+/-15%) to SNS. Similar results were seen with the cyclic GMP analogue, 8-Br-cGMP (0.5 mM, n=12).. Our results indicate that NO and sulphonlyurea-sensitive channels act in a complementary fashion, but appear to be independent of each other in the regulation of HR during cardiac SNS activation.

Topics: Analysis of Variance; Animals; Cyclic GMP; Diazoxide; Electric Stimulation; Enzyme Inhibitors; Glyburide; Guinea Pigs; Heart Atria; Heart Rate; Hypoxia; In Vitro Techniques; Male; Nitric Oxide; Norepinephrine; Potassium Channel Blockers; Potassium Channels; Stellate Ganglion; Sympathetic Nervous System; Tolbutamide

N(G)-monomethyl-L-arginine (L-NMMA) has been reported to be elevated in uremic patients. Based on the hypothesis that the pathogenesis of the anemia of renal disease might be due to the perturbation of transcription factors of the erythropoietin (Epo) gene by L-NMMA, the present study was designed to investigate the effect of L-NMMA on Epo gene expression through the GATA transcription factor. L-NMMA caused decreased levels of NO, cyclic guanosine monophosphate (cGMP), and Epo protein in Hep3B cells. L-NAME (analogue of L-NMMA) also inhibited Epo production in anemic mice. Transfection of the Epo promoter-luciferase gene into Hep3B cells revealed that L-NMMA inhibited the Epo promoter activity. However, L-NMMA did not inhibit the Epo promoter activity when mutated Epo promoter (GATA to TATA) was transfected, and L-NMMA did not affect the enhancer activity. Electrophoretic mobility shift assays demonstrated the stimulation of GATA binding activity by L-NMMA. However, L-NMMA had no effect on the binding activity of hepatic nuclear factor-4, COUP-TF1, hypoxia-inducing factor-1, or NF-kappaB. Furthermore, cGMP inhibited the L-NMMA-induced GATA binding activity. L-NMMA also increased GATA-2 messenger RNA expression. These results demonstrate that L-NMMA suppresses Epo gene expression by up-regulation of the GATA transcription factor and support the hypothesis that L-NMMA is one of the candidate substances that underlie the pathogenesis of renal anemia. (Blood. 2000;96:1716-1722)

Topics: Animals; Base Sequence; Binding Sites; Cyclic GMP; DNA; DNA-Binding Proteins; Enzyme Inhibitors; Erythropoietin; GATA2 Transcription Factor; Gene Expression Regulation; Humans; Hypoxia; Luciferases; Mice; Molecular Sequence Data; Mutation; NG-Nitroarginine Methyl Ester; Nitric Oxide; omega-N-Methylarginine; Promoter Regions, Genetic; Protein Binding; Recombinant Fusion Proteins; Transcription Factors; Tumor Cells, Cultured

Single intraperitoneal injection of melatonin in a dose of 1 mg/kg prevented accumulation of cGMP and intensification of lipid peroxidation in the hippocampus and habenula of rats exposed to acute hypobaric hypoxia (12,000 m). Changes in habenular content of cGMP suggest that melatonin prevents hypoxia-induced activation of heme-oxygenase.

Topics: Acute Disease; Animals; Cyclic AMP; Cyclic GMP; Enzyme Activation; Habenula; Heme Oxygenase (Decyclizing); Hippocampus; Hypoxia; Lipid Peroxidation; Male; Malondialdehyde; Melatonin; Rats

Since recent studies show that pial artery dilation during a 20 or 40 min hypoxic exposure was less than that observed during a 5 or 10 min exposure, stimulus duration determines the nature of the vascular response to hypoxia. Decremented hypoxic pial dilation during longer exposure periods results, at least in part, from decreased release of methionine enkephalin (Met), an opioid known to contribute to dilation during hypoxia. Nitric oxide and cGMP contribute to both release and the vascular response to this opioid. The present study was designed to determine if the stimulus duration modulates the interaction between opioids and NO in hypoxic pial dilation using newborn pigs equipped with a closed cranial window. Elevation of CSF cGMP during hypoxia (Po2 approximately 35 mmHg) was dependent on stimulus duration (435+/-31, 934+/-46, 747+/-25, and 623+/-17 fmol/ml cGMP during normoxia and after 10, 20, and 40 min of hypoxia). Met-induced pial dilation during hypoxia was also stimulus duration dependent (7+/-1, 10+/-1, and 15+/-1, vs. 4+/-1, 6+/-1, and 8+/-2 vs. 2+/-1, 3+/-1, and 5+/-1% for 10(-10), 10(-8), 10(-6) M Met during normox and after 20, and 40 min of hypoxia). Additionally, the release of cGMP by Met during hypoxia was also stimulus duration dependent (1.8+/-0.1 vs. 1.6+/-0.1 vs. 1.3+/-0.1 fold change in CSF cGMP for 10(-8) M Met during normoxia and after 20 and 40 min of hypoxia). These data indicate that the diminished role of Met in pial dilation during longer hypoxic exposure periods results from a diminished capacity of this opioid to elicit dilation. Such impaired dilation is correlated with diminished stimulated cGMP release. These data also suggest that diminished CSF cGMP release during prolonged hypoxia contributes to decreased release of Met during longer hypoxic periods. Therefore, stimulus duration modulates the interaction between opioids and NO in hypoxic pial artery dilation.

Topics: Animals; Animals, Newborn; Blood Gas Analysis; Blood Pressure; Cerebral Arteries; Cyclic GMP; Enkephalin, Methionine; Female; Hydrogen-Ion Concentration; Hypoxia; Hypoxia, Brain; Male; Nitric Oxide; Nitroarginine; Pia Mater; Swine; Vasodilation

Nitric oxide (NO) functions as an endothelium-derived relaxation factor and regulates vascular resistance. Recent studies in this laboratory(Arch.Biochem.Biophys.323, 27-32, 1995) revealed that the lifetime of NO significantly increased at physiologically low levels of oxygen concentrations and, hence, this gaseous radical strongly inhibited mitochondrial electron transport for a fairly long duration at low oxygen concentrations. The present work describes the effect of oxygen concentration on NO-induced relaxation and guanylate cyclase (GC) activity of endothelium-denuded aorta of the rat. Both NO and 2,2 '-(hydroxynitrosohydrazono)bis-ethanamine (NOC18), an NO donor, induced the relaxation of endothelium-denuded helical segments of rat aorta which were contracted by norepinephrine. NO-dependent relaxation of arterial specimens was enhanced by lowering oxygen concentration in the medium with concomitant increase in their cGMP levels. Anoxia induced the relaxation of the aorta by some NO-enhanceable and methylene blue-insensitive mechanism. These results suggested that local concentrations of oxygen might play important roles in the regulation of NO-dependent GC activity and vascular tonus of resistance arteries.

Topics: Animals; Aorta, Thoracic; Cyclic GMP; Endothelium, Vascular; Guanylate Cyclase; Hypoxia; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Norepinephrine; Oxygen; Rats; Rats, Wistar

The objective of this study was to investigate the effect of L-arginine supplementation on myocardial cell death secondary to hypoxia-reoxygenation. Isolated rat hearts (n = 51) subjected to 40 min of hypoxia and 90 min of reoxygenation received 3 mM L-arginine and/or 1 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; a selective inhibitor of soluble guanylyl cyclase) throughout the experiment or during the equilibration, hypoxia, or reoxygenation periods. The incorporation of L-[3H]arginine into myocytes during energy deprivation was investigated in isolated adult rat myocytes. The addition of L-arginine to the perfusate throughout the experiment resulted in higher cGMP release (P < 0.05), reduced lactate dehydrogenase release (P < 0.05), and increased pressure-rate product (P < 0.05) during reoxygenation. These effects were reproduced when L-arginine was added only during equilibration, but addition of L-arginine during hypoxia or reoxygenation had no effect. Addition of ODQ either throughout the experiment or only during reoxygenation reversed the beneficial effects of L-arginine. L-[3H]arginine was not significantly incorporated into isolated myocytes subjected to energy deprivation. We conclude that L-arginine supplementation protects the myocardium against reoxygenation injury by cGMP-mediated actions. To be effective during reoxygenation, L-arginine must be added before anoxia.

Topics: Animals; Arginine; Cell Death; Cells, Cultured; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; Hypoxia; L-Lactate Dehydrogenase; Male; Muscle Fibers, Skeletal; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Nitric Oxide; Oxadiazoles; Oxygen Consumption; Quinoxalines; Rats; Rats, Sprague-Dawley

The effects of transfer of the endothelial nitric oxide synthase (eNOS) gene to the lung were studied in mice. After intratracheal administration of AdCMVbetagal, expression of the beta-galactosidase reporter gene was detected in pulmonary airway cells, in alveolar cells, and in small pulmonary arteries. Gene expression with AdCMVbetagal peaked 1 day after administration and decayed over a 7- to 14-day period, whereas gene expression after AdRSVbetagal transfection peaked on day 5 and was sustained over a 21- to 28-day period. One day after administration of AdCMVeNOS, eNOS protein levels were increased, and there was a small reduction in mean pulmonary arterial pressure and pulmonary vascular resistance. The pressure-flow relationship in the pulmonary vascular bed was shifted to the right in animals transfected with eNOS, and pulmonary vasodepressor responses to bradykinin and the type V cGMP-selective phosphodiesterase inhibitor zaprinast were enhanced, whereas systemic responses were not altered. Pulmonary vasopressor responses to endothelin-1 (ET-1), angiotensin II, and ventilatory hypoxia were reduced significantly in animals transfected with the eNOS gene, whereas pressor responses to norepinephrine and U46619 were not changed. Systemic pressor responses to ET-1 and angiotensin II were similar in eNOS-transfected mice and in control mice. Intratracheal administration of AdRSVeNOS attenuated the increase in pulmonary arterial pressure in mice exposed to the fibrogenic anticancer agent bleomycin. These data suggest that transfer of the eNOS gene in vivo can selectively reduce pulmonary vascular resistance and pulmonary pressor responses to ET-1, angiotensin II, and hypoxia; enhance pulmonary depressor responses; and attenuate pulmonary hypertension induced by bleomycin. Moreover, these data suggest that in vivo gene transfer may be a useful therapeutic intervention for the treatment of pulmonary hypertensive disorders.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenoviridae; Angiotensin II; Animals; Antimetabolites, Antineoplastic; beta-Galactosidase; Bleomycin; Blood Flow Velocity; Blood Pressure; Bradykinin; Cyclic GMP; Endothelin-1; Gene Transfer Techniques; Genes, Reporter; Hypertension, Pulmonary; Hypoxia; Mice; Mice, Inbred Strains; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Norepinephrine; Phosphodiesterase Inhibitors; Pulmonary Alveoli; Pulmonary Artery; Pulmonary Circulation; Pulmonary Wedge Pressure; Purinones; Sympathomimetics; Vasoconstrictor Agents

These studies tested whether fetal artery reactivity is sensitive to both acute changes in oxygen levels (in vitro) and chronic changes (in utero).. Pregnant guinea pigs near term were exposed to either normoxia or hypoxia (12% oxygen) for 4 or 7 days. The effect of decreasing PO (2 ) in vitro (acute hypoxia) on relaxation in response to acetylcholine, A23187, sodium nitroprusside, and 8-bromo-cyclic guanosine monophosphate was measured in isolated carotid arteries from normoxic fetuses. In separate experiments relaxation in response to acetylcholine and sodium nitroprusside of endothelially intact and denuded fetal arteries from fetuses exposed to normoxic conditions and long-term (4 and 7 days) hypoxic conditions was measured in the presence and absence of nitro-L -arginine (10(-4) mol/L).. Acute hypoxia inhibited endothelium-dependent relaxation in response to acetylcholine and A23187, increased sensitivity to sodium nitroprusside, but had no effect on relaxation in response to 8-bromo-cyclic guanosine monophosphate. Chronic hypoxia (4 but not 7 days) inhibited maximal relaxation of arteries in response to acetylcholine but not relaxation of arteries in response to sodium nitroprusside with respect to relaxation seen in arteries from normoxic fetuses. Nitro-L -arginine attenuated the differences between normoxic and hypoxic fetuses in acetylcholine response.. Hypoxia may alter relaxation of fetal arteries by decreasing the availability of oxygen for nitric oxide production and causing vascular adaptations related to altered nitric oxide release.

Topics: Acetylcholine; Acute Disease; Animals; Arteries; Calcimycin; Chronic Disease; Cyclic GMP; Disease Models, Animal; Female; Guinea Pigs; Hypoxia; Muscle, Smooth, Vascular; Nitric Oxide; Nitroprusside; Pregnancy; Vasodilator Agents

Chronic exposure to hypoxia from birth increased the tolerance of the rabbit heart to subsequent ischemia compared with age-matched normoxic controls. The nitric oxide donor GSNO increased recovery of post-ischemic function in normoxic hearts to values not different from hypoxic controls, but had no effect on hypoxic hearts. The nitric oxide synthase inhibitors L-NAME and L-NMA abolished the cardioprotective effect of hypoxia. Message and catalytic activity for constitutive nitric oxide synthase as well as nitrite, nitrate, and cGMP levels were elevated in hypoxic hearts. Inducible nitric oxide synthase was not detected in normoxic or chronically hypoxic hearts. Increased tolerance to ischemia in rabbit hearts adapted to chronic hypoxia is associated with increased expression of constitutive nitric oxide synthase.

Topics: Adaptation, Physiological; Animals; Chronic Disease; Cyclic GMP; Hypoxia; Myocardial Ischemia; Myocardium; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitrites; Rabbits; RNA, Messenger

The purpose of this study was to determine whether E-4010, a newly synthesized potent and selective orally active phosphodiesterase (PDE) 5 inhibitor, would prevent the development of chronic hypoxia-induced pulmonary hypertension in rats. In conscious, pulmonary hypertensive rats, a single oral administration of E-4010 (1.0 mg/kg) caused an acute, long-lasting reduction in mean pulmonary arterial pressure (PAP), with no significant effects on systemic arterial pressure, cardiac output, and heart rate. In rats that received food containing 0.01 or 0.1% E-4010 during the 3-wk exposure to hypoxia, mean PAP was significantly decreased (mean PAP 24.0 +/- 0.9, 16.2 +/- 0.8, and 12.8 +/- 0.5 mmHg in rats treated with 0, 0.01, and 0.1% E-4010-containing food, respectively), whereas mean systemic arterial pressure was unchanged and cardiac output was slightly increased compared with chronically hypoxic control rats. Right ventricular hypertrophy, medial wall thickness in pulmonary arteries corresponding to the respiratory and terminal bronchioles, and the degree of muscularization of more distal arteries were less severe in E-4010-treated rats. Long-term treatment with E-4010 caused an increase in cGMP levels in lung tissue and plasma but not in aortic tissue and no significant change in cAMP levels in either lung, aorta, or plasma. These results suggest that long-term oral treatment with E-4010 reduced the increase in PAP, right ventricular hypertrophy, and pulmonary arterial remodeling induced by exposure to chronic hypoxia, probably through increasing cGMP levels in the pulmonary vascular smooth muscle.

Topics: Animals; Aorta; Cyclic GMP; Hemodynamics; Hypertension, Pulmonary; Hypoxia; Lung; Male; Nitriles; Phosphodiesterase Inhibitors; Piperidines; Rats; Rats, Sprague-Dawley; Time Factors

The management of sick newborn infants who have sustained a hypoxic insult is a common clinical problem but relatively little is known about the recovery process. The aim of this study was to investigate this process in newborn piglets.. Thirty five newborn piglets were exposed to chronic hypobaric hypoxia for three days, either from birth, three or 14 days of age, and were allowed to recover for one, three, or six days. Control animals of relevant age were also studied. The heart weight ratio and pulmonary arterial muscularity were measured. Endothelial dependent and independent relaxation of the isolated intrapulmonary conduit arteries was determined in classical organ chamber studies, together with measurement of basal and stimulated cGMP accumulation.. After six days of recovery the hypoxia induced right ventricular hypertrophy and pulmonary arterial medial hypertrophy had decreased in all animals but values were still abnormal in the two younger age groups. Relaxation was still impaired during the first three days of recovery in all groups, had normalised by six days in the two youngest groups, but relaxation (both endothelium dependent and independent) remained impaired in older animals. In these older animals basal nitric oxide (NO) production and basal and stimulated cGMP accumulation was normal.. The recovery of the smooth muscle cells lags behind that of the endothelial cells. A normal stimulated increase in cGMP with reduced relaxation suggests an altered threshold for cGMP effected relaxation. These findings help to explain why some hypoxic infants require protracted NO therapy.

Topics: Acetylcholine; Animals; Animals, Newborn; Calcimycin; Cyclic GMP; Enzyme Inhibitors; Hypertension, Pulmonary; Hypertrophy; Hypertrophy, Right Ventricular; Hypoxia; Nitric Oxide; omega-N-Methylarginine; Phosphodiesterase Inhibitors; Pulmonary Artery; Purinones; Swine; Tunica Intima; Vasoconstriction; Vasodilator Agents

Pulmonary vascular resistance falls rapidly after birth, but endothelium-dependent relaxation is relatively poor during the perinatal period. Atrial natriuretic peptide (ANP) is a potent vasodilator; however, its role in the process of perinatal adaptation is uncertain. Porcine intrapulmonary conduit arteries (IPA) from fetal, newborn (< 5 min), 3-, 6-, and 17-d-old, and adult pigs, and from piglets made hypoxic from 0 to 3, 3 to 6, or 14 to 17 d, were isolated and mounted for isometric force recording. Rings were precontracted with prostaglandin-F2 alpha (PGF2 alpha, 10 microM) or KCl (40 mM). ANP was added cumulatively (10 pM to 100 nM). C-type natriuretic peptide (CNP) was added as a single concentration of 100 nM. Accumulation of cGMP under basal conditions and stimulated by ANP or CNP was measured by radioimmunoassay system. Frozen sections of lung tissue were incubated with 125I-labeled alpha-ANP, and binding site density was assessed on IPA with an image analysis system. ANP relaxed IPA in pigs at all ages, but the effect was significantly greater at 6 and 17 d of age. Hypoxia in animals from 14 to 17 d old impaired ANP-induced relaxation. CNP relaxed IPA poorly: < 12% at all ages. ANP increased cGMP accumulation in both normal and hypoxic animals. CNP did not increase cGMP generation in IPA from normal animals but did so in IPA from 3-d-old hypoxic animals. ANP-specific binding sites were demonstrated on the pulmonary artery smooth muscle cells, with greater binding in the young animals. The increased relaxant responses to ANP during adaptation may be important in maintaining low pulmonary vascular resistance. In contrast, CNP was largely ineffective in relaxing pulmonary arteries.

Topics: Age Factors; Animals; Animals, Newborn; Atrial Natriuretic Factor; Binding Sites; Cyclic GMP; Fetus; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Natriuretic Peptide, C-Type; Pulmonary Artery; Sus scrofa; Vascular Resistance; Vasodilator Agents

Inhaled nitric oxide (NO) may downregulate the endogenous NO/cyclic guanosine monophosphate (cGMP) pathway, potentially explaining clinical rebound pulmonary hypertension. We determined if inhaled NO decreases pulmonary cGMP levels, if the possible down-regulation is the same as with nifedipine, and if regulation also occurs with the cyclic adenosine monophosphate (cAMP) pathway. Rats were exposed to 3 wk of normoxia, hypoxia (10% O2), or monocrotaline (MCT; single dose = 60 mg/kg) and treated with either nothing (control), inhaled NO (20 ppm), or nifedipine (10 mg x kg(-1) x day(-1). The lungs were then isolated and perfused with physiologic saline. Perfusate cGMP, prostacyclin, and cAMP levels were measured. Perfusate cGMP was not altered by inhaled NO or nifedipine in normoxic or MCT rats. Although hypoxia significantly increased cGMP by 128%, both inhaled NO and nifedipine equally prevented the hypoxic increase. Inhibition of the NO/cGMP pathway with N(G)-nitro-L-arginine methyl ester (L-NAME) decreased cGMP by 72% and 88% in normoxic and hypoxic lungs. Prostacyclin and cAMP levels were not altered by inhaled NO or nifedipine. L-NAME significantly decreased cGMP levels, whereas inhaled NO had no effect on cGMP in normoxic or MCT lungs, suggesting that inhaled NO does not inhibit the NO/cGMP pathway. Inhaled NO decreased cGMP in hypoxic lungs, however, nifedipine had the same effect, which indicates the decrease is not specific to inhaled NO.. High pulmonary pressure after discontinuation of inhaled nitric oxide (NO) may be secondary to a decrease in the natural endogenous NO vasodilator. This rat study suggests that inhaled NO either does not alter endogenous NO or that it has similar effects as nifedipine.

Topics: 6-Ketoprostaglandin F1 alpha; Administration, Inhalation; Animals; Bronchodilator Agents; Cyclic AMP; Cyclic GMP; Down-Regulation; Enzyme Inhibitors; Epoprostenol; Hypertension, Pulmonary; Hypoxia; Lung; Male; Monocrotaline; NG-Nitroarginine Methyl Ester; Nifedipine; Nitric Oxide; Poisons; Rats; Rats, Sprague-Dawley; Vasodilator Agents

Decreased oxygen delivery to cells (hypoxia) is prevalent in a number of important diseases. Little is known about mechanisms of oxygen sensing at the cellular level or about whether functional correlates of oxygen sensing exist. In this study, we examined the impact of hypoxia on stimulated epithelial ion transport function. T84 cells, a model of intestinal epithelia, were grown on permeable supports, exposed to hypoxia (range 1-21% O2) for periods of time between 0 and 72 h and assessed for stimulated ion transport. Hypoxia evoked a specific decrease in cyclic nucleotide-stimulated (cAMP and cGMP) but not Ca++-stimulated ion transport. 86Rb (K+ tracer) uptake and 125I (Cl- tracer) efflux were reduced in hypoxic cells by >50% and >40%, respectively, fluid movement was reduced by hypoxia (>50% decrease) and reoxygenation resulted in partial recovery of the ion transport responses. Stimulated and basal levels of both cAMP and cGMP were decreased in response to hypoxia, although intracellular ATP levels were unaltered under similar conditions. Exogenous addition of cobalt, nickel or manganese, all of which compete for oxygen binding on heme-containing proteins, mimicked hypoxia. Because guanylate cyclase is a heme protein, we measured the influence of cobalt on activity of guanylate cyclase in purified plasma membrane preparations and found cobalt to inhibit stimulated cGMP levels in this cell-free system. Finally, pharmacological lowering of intracellular cGMP (using LY83583) resulted in decreased cAMP-stimulated Cl- secretion, and direct elevation of cGMP (using 8-bromo-cGMP or dibutyryl-cGMP) restored this hypoxia-induced activity. We conclude that a potential oxygen-sensing mechanism of epithelial cells involves the cooperation of heme-containing proteins such as guanylate cyclase and that biochemical cross-talk between cAMP- and cGMP-stimulated pathways may be important in such responses.

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Biological Transport; Cell Line; Cyclic AMP; Cyclic GMP; Down-Regulation; Epithelial Cells; Guanylate Cyclase; Heme; Humans; Hypoxia; Intestinal Mucosa; Nucleotides, Cyclic; Oxygen; Water-Electrolyte Balance

This study was designed to investigate the potential role of endogenous peroxynitrite (ONOO-) formation in the inhibition of cardiac muscle contractility and mitochondrial respiration during posthypoxic reoxygenation. Isometric contraction of isolated rat left ventricular posterior papillary muscle was virtually eliminated at the end of an exposure to 15 minutes of hypoxia and remained 40+/-5% depressed an hour after the reintroduction of O2. O2 uptake by rat left ventricular cardiac muscle, measured by a Clark-type O2 electrode, was also inhibited by 24+/-2% at 10 minutes after reoxygenation. The inhibition of contractility and respiration during posthypoxic reoxygenation was markedly attenuated by the NO synthase inhibitor nitro-L-arginine, exogenous superoxide dismutase, and the ONOO- scavenger urate but not by the hydroxyl radical scavenger mannitol. Generation of ONOO- with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) plus the superoxide-releasing agent pyrogallol caused an irreversible inhibition of cardiac contractile and respiratory function. Unlike ONOO-, exogenous (SNAP) and endogenous (bradykinin) sources of NO inhibited contractility in a reversible manner. Under conditions of comparable amounts of respiratory inhibition in unstimulated incubated muscle, the NO-dependent agents and the mitochondrial antagonist NaCN produced a smaller degree of suppression of contractility compared with ONOO- and posthypoxic reoxygenation. These results are consistent with a contributing role for endogenous ONOO- formation in the inhibition of cardiac muscle contractility and mitochondrial respiration during posthypoxic reoxygenation.

Topics: Animals; Bradykinin; Cyclic GMP; Free Radical Scavengers; Hydroxyl Radical; Hypoxia; In Vitro Techniques; Kinetics; Mannitol; Mitochondria, Heart; Myocardial Contraction; Nitrates; Nitric Oxide; Nitroarginine; Oxidants; Oxygen Consumption; Penicillamine; Rats; S-Nitroso-N-Acetylpenicillamine; Sodium Cyanide; Superoxide Dismutase

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and blood vessel remodeling. Its expression is up-regulated in vascular smooth muscle cells by a number of conditions, including hypoxia. Hypoxia increases the transcriptional rate of VEGF via a 28-base pair enhancer located in the 5'-upstream region of the gene. The gas molecules nitric oxide (NO) and carbon monoxide (CO) are important vasodilating agents. We report here that these biological molecules can suppress the hypoxia-induced production of VEGF mRNA and protein in smooth muscle cells. In transient expression studies, both NO and CO inhibited the ability of the hypoxic enhancer we have previously identified to activate gene transcription. Furthermore, electrophoretic mobility shift assays indicated decreased binding of hypoxia-inducible factor 1 (HIF-1) to this enhancer by nuclear proteins isolated from CO-treated cells, although HIF-1 protein levels were unaffected by CO. Given that both CO and NO activate guanylyl cyclase to produce cGMP and that a cGMP analog (8-Br-cGMP) showed a similar suppressive effect on the hypoxic induction of the VEGF enhancer, we speculate that the suppression of VEGF by these two gas molecules occurs via a cyclic GMP-mediated pathway.

Topics: Animals; Carbon Monoxide; Cattle; Cells, Cultured; Cyclic GMP; DNA-Binding Proteins; Endothelial Growth Factors; Enhancer Elements, Genetic; Gene Expression Regulation; Guanylate Cyclase; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Lymphokines; Muscle, Smooth, Vascular; Nitric Oxide; Nuclear Proteins; RNA, Messenger; Transcription Factors; Transcription, Genetic; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Vasodilator Agents

We studied the role of nitric oxide (NO) on the regulation of blood flow in the immature kidney during hypoxia, resuscitation and the recovery period using the NO inhibitor N(omega)-nitro-L-arginine (L-NNA) in a newborn piglet model, and the possibility of urinary cGMP as an index of renal function. After administration of L-NNA, the blood flow in both the cortex and medulla significantly decreased, indicating that NO is constantly released to maintain renal circulation. During hypoxia, the renal blood flow fell remarkably, and there were no differences between the control and L-NNA groups. During the post-resuscitation period, the recovery of renal blood flow was significantly suppressed in L-NNA administration, and it was speculated that NO might be an important factor for recovery of circulation from vasoconstriction due to hypoxemia. Urinary cGMP/cr was significantly increased on recovery from hypoxemia and was suppressed by L-NNA administration. This result suggested that the change in cGMP/cr represents renal blood flow change. We conclude that NO may play an important role in maintaining basal hemodynamics, and may also be a crucial factor for recovery from post-hypoxic vasoconstriction in premature kidneys. Urinary cGMP/cr might serve as one of the indices for assessment of premature renal circulation.

Topics: Animals; Animals, Newborn; Cyclic GMP; Enzyme Inhibitors; Hypoxia; Nitric Oxide; Nitroarginine; Reference Values; Renal Circulation; Swine

Although nitric oxide (NO) and calcium sensitive K+ channel (Kca) activation contribute to hypoxic pial artery dilation in the piglet, responses to the NO releasers SNP and SNAP are unchanged by the Kca channel antagonist iberiotoxin. These data suggest that NO does not elicit dilation via Kca channel activation. The present study was designed to determine if dilation by Kca channel activation is mediated by NO in newborn pigs equipped with a closed cranial window. NS1619 (10(-8), 10(-6) M), a Kca agonist, produced dilation that was unchanged by the NO synthase inhibitor, L-NNA (10(-6) or 10(-3) M) (11+/-1 and 20+/-1 vs. 11+/-1 and 18+/-1% before and after L-NNA 10(-3) M). NS1619 dilation also was not associated with increased CSF cGMP and was unchanged by Rp 8-Bromo cGMPs, a cGMP antagonist (9+/-1 and 17+/-1 vs. 9+/-1 and 16+/-2% before and after Rp 8-Bromo cGMPs 10(-5) M). Iberiotoxin (10(-7) M) attenuated hypoxic dilation but hypoxia associated CSF cGMP release was unchanged (418+/-11 and 897+/-31 vs. 419+/-10 and 896+/-25 fmol/ml for control and moderate hypoxia before and after iberiotoxin). Coadministration of L-NNA with iberiotoxin further decremented hypoxic pial dilation and blocked the hypoxia-associated rise in CSF cGMP. These data show that pial artery dilation by Kca channel activation is not mediated by NO/cGMP. Further, these data suggest that NO and the Kca channel act at different sites in their contributions to hypoxic pial artery dilation.

Topics: Animals; Arteries; Benzimidazoles; Cerebrovascular Circulation; Cyclic GMP; Endorphins; Enzyme Inhibitors; Female; Hypoxia; Intermediate-Conductance Calcium-Activated Potassium Channels; Male; Nitric Oxide; Nitroarginine; Pia Mater; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Calcium-Activated; Swine; Vasodilation

1. The role of endogenous nitric oxide (NO) generated by neuronal nitric oxide synthase (NOS-1) in the control of respiration during hypoxia and hypercapnia was assessed using mutant mice deficient in NOS-1. 2. Experiments were performed on awake and anaesthetized mutant and wild-type control mice. Respiratory responses to varying levels of inspired oxygen (100, 21 and 12% O2) and carbon dioxide (3 and 5% CO2 balanced oxygen) were analysed. In awake animals, respiration was monitored by body plethysmograph along with oxygen consumption (VO2), CO2 production (VCO2) and body temperature. In anaesthetized, spontaneously breathing mice, integrated efferent phrenic nerve activity was monitored as an index of neural respiration along with arterial blood pressure and blood gases. Cyclic 3',5'-guanosine monophosphate (cGMP) levels in the brainstem were analysed by radioimmunoassay as an index of nitric oxide generation. 3. Unanaesthetized mutant mice exhibited greater respiratory responses during 21 and 12% O2 than the wild-type controls. Respiratory responses were associated with significant decreases in oxygen consumption in both groups of mice, and the magnitude of change was greater in mutant than wild-type mice. Changes in CO2 production and body temperature, however, were comparable between both groups of mice. 4. Similar augmentation of respiratory responses during hypoxia was also observed in anaesthetized mutant mice. In addition, five of the fourteen mutant mice displayed periodic oscillations in respiration (brief episodes of increases in respiratory rate and tidal phrenic nerve activity) while breathing 21 and 12% O2, but not during 100% O2. The time interval between the episodes decreased by reducing inspired oxygen from 21 to 12% O2. 5. Changes in arterial blood pressure and arterial blood gases were comparable at any given level of inspired oxygen between both groups of mice, indicating that changes in these variables do not account for the differences in the response to hypoxia. 6. Respiratory responses to brief hyperoxia (Dejours test) and to cyanide, a potent chemoreceptor stimulant, were more pronounced in mutant mice, suggesting augmented peripheral chemoreceptor sensitivity. 7. cGMP levels were elevated in the brainstem during 21 and 12% O2 in wild-type but not in mutant mice, indicating decreased formation of nitric oxide in mutant mice. 8. The magnitude of respiratory responses to hypercapnia (3 and 5% CO2 balanced oxygen) was comparable in

Topics: Animals; Blood Pressure; Body Temperature; Brain Stem; Carbon Dioxide; Chemoreceptor Cells; Crosses, Genetic; Cyclic GMP; Hypoxia; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Mutant Strains; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Oxygen; Oxygen Consumption; Phrenic Nerve; Respiration

Nitric oxide (NO), opioids, and ATP-sensitive K+ (KATP) channel activation contribute to hypoxia-induced pial artery dilation. NO releasers and cGMP analogs increase opioid concentration in cerebrospinal fluid (CSF) and elicit dilation via KATP channel activation. Opioids themselves also elicit dilation via KATP channel activation. This study was designed to investigate the relationships among the above mechanisms in hypoxic pial artery dilation using newborn pigs equipped with a closed cranial window. Cromakalim (10(-8) and 10(-6) M), a KATP agonist, produced dilation that was unchanged by the NO synthase inhibitor N-nitro-L-arginine (L-NNA, 10(-6) and 10(-3) M): 13 +/- 1 and 31 +/- 1 vs. 14 +/- 1 and 31 +/- 1% before and after 10(-3) M L-NNA. Cromakalim dilation also was not associated with increased CSF cGMP and was unchanged by the Rp diastereomer of 8-bromoguanosine 3',5'-cyclic monophosphothioate, a cGMP antagonist. Glibenclamide (10(-6) M), a KATP antagonist, attenuated hypoxic dilation but hypoxia-associated CSF cGMP release was unchanged: 457 +/- 12 and 935 +/- 30 vs. 458 +/- 11 and 921 +/- 22 fmol/ml. Coadministration of L-NNA with glibenclamide had no further effect on the already diminished hypoxic dilation but blocked the hypoxia-associated rise in CSF cGMP. Cromakalim had no effect on CSF methionine enkephalin: 1,012 +/- 28 and 1,062 +/- 32 pg/ml. These data show that KATP channel agonists do not elicit dilation via NO/cGMP and do not release opioids. NO release during hypoxia also is independent of KATP channel activation. These data suggest that hypoxic dilation results from the sequential release of NO, cGMP, and opioids, which in turn activate the KATP channel.

Topics: Adenosine Triphosphate; Animals; Arteries; Blood Pressure; Calcitonin Gene-Related Peptide; Cromakalim; Cyclic GMP; Female; Glyburide; Hypoxia; Male; Nitric Oxide; Opioid Peptides; Pia Mater; Potassium Channels; Swine; Vasodilation; Vasodilator Agents

Nitric oxide (NO) modulates the endogenous NO-cGMP pathway. We determined whether prolonged inhaled NO downregulates the NO-cGMP pathway, which may explain clinically observed rebound pulmonary hypertension. Rats were placed in a normoxic (N; 21% O2) or hypoxic (H; 10% O2) environment with and without inhaled NO (20 parts/million) for 1 or 3 wk. Subsequently, nitric oxide synthase (NOS) and soluble guanylate cyclase (GC) activity and endothelial NOS (eNOS) protein levels were measured. Perfusate cGMP levels and endothelium-dependent and -independent vasodilation were determined in isolated lungs. eNOS protein levels and NOS activity were not altered by inhaled NO in N or H rats. GC activity was decreased by 60 +/- 10 and 55 +/- 11% in N and H rats, respectively, after 1 wk of inhaled NO but was not affected after 3 wk. Inhaled NO had no effect on perfusate cGMP in N lungs. Inhaled NO attenuated the increase in cGMP levels caused by 3 wk of H by 57 +/- 11%, but there was no rebound in cGMP after 24 h of recovery. Endothelium-dependent vasodilation was not altered, and endothelium-independent vasodilation was not altered (N) or slightly increased (H, 10 +/- 3%) by prolonged inhaled NO. In conclusion, inhaled NO did not alter the endogenous NO-cGMP pathway as determined by eNOS protein levels, NOS activity, or endothelium-dependent vasodilation under N and H conditions. GC activity was decreased after 1 wk; however, GC activity was not altered by 3 wk of inhaled NO and endothelium-independent vasodilation was not decreased.

Topics: Administration, Inhalation; Animals; Blotting, Western; Chromatography, Gas; Cyclic GMP; Endothelium, Vascular; Guanylate Cyclase; Hypoxia; In Vitro Techniques; Male; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Rats; Rats, Sprague-Dawley; Signal Transduction; Vasodilation

Inhaled nitric oxide (iNO) causes selective pulmonary vasodilation by increasing pulmonary vascular levels of cyclic guanosine monophosphate (cGMP). Dipyridamole, a drug with several putative vasodilator mechanisms, is an inhibitor of cGMP-specific phosphodiesterases (PDE5); it therefore has the potential to increase pulmonary vascular cGMP levels, lower pulmonary vascular resistance, augment iNO-induced pulmonary vasodilation, and attenuate excessive pulmonary vasoreactivity. To test dipyridamole in the pulmonary circulation, we studied pediatric patients undergoing cardiac catheterization who had severe resting pulmonary hypertension (Group 1; n = 11) or exaggerated acute hypoxia-induced pulmonary vasoconstriction (Group 2; n = 4). In Group 1, we compared the effects of iNO (20 ppm), dipyridamole (0.6 mg/kg), and combined treatments (iNO + dipyridamole) on pulmonary and systemic hemodynamics. In Group 2 we measured the pulmonary and systemic effects of dipyridamole while the patients were breathing room air and hypoxic gas mixtures (FIO2 = 0.16). One patient in Group 1 had a hypotensive response to dipyridamole and was exluded from study. In the remaining 12 studies done on 10 patients, iNO caused a selective decrease in mean pulmonary artery pressure (Ppa) and indexed pulmonary vascular resistance (PVRI) without affecting mean aortic pressure (Pao) or indexed systemic vascular resistance (SVRI). Dipyridamole decreased PVRI to similar values as did iNO, but this effect was primarily due to an increase in cardiac index (CI), and was not associated with any change in Ppa, and was associated with a decrease in Pao and SVRI. In comparison with individual treatments, combined therapy (iNO + dipyridamole) did not augment pulmonary vasodilation in the group as a whole; however, in 50% of patients, combined therapy decreased PVRI by 20% more than did iNO or dipyridamole alone. In Group 2, Ppa and the pulmonary-to-systemic resistance ratio (Rp/Rs) increased to suprasystemic levels during acute hypoxia. Pretreatment with dipyridamole blunted the increase in Ppa and Rp/Rs during repeat hypoxia, keeping Ppa at a subsystemic level and Rp/Rs < 1. We conclude that: (1) dipyridamole nonselectively reduces PVRI, primarily through an increase in CI; (2) in combination with iNO, dipyridamole augments the decrease in PVRI in some patients; and (3) dipyridamole blunts the severity of acute hypoxic pulmonary vasoconstriction in children with exaggerated hypoxic pressor respo

Topics: Administration, Inhalation; Adolescent; Adult; Aorta; Blood Pressure; Bronchodilator Agents; Cardiac Catheterization; Cardiac Output; Child; Child, Preschool; Cyclic GMP; Dipyridamole; Female; Humans; Hypertension, Pulmonary; Hypoxia; Infant; Male; Nitric Oxide; Phosphodiesterase Inhibitors; Pulmonary Artery; Pulmonary Circulation; Vascular Resistance; Vasodilator Agents

1. Cyclic guanosine 3'-5'-monophosphate (cyclic GMP) is the second messenger of important physiologically active mediators controlling the pulmonary vascular tone. To potentiate the effects of cyclic GMP on the pulmonary vasculature, we used DMPPO, a new selective PDE-5 inhibitor, and examined its action in a rat model of hypoxic pulmonary hypertension. 2. Levels of cyclic GMP measured during baseline conditions at 5 and 60 min of perfusion were similar in the perfusate of isolated lungs from normoxic and chronically hypoxic rats and did not differ with time. Pretreatment with DMPPO (1 microM) induced a larger increase in cyclic GMP concentration in the perfusate from chronically hypoxic rat lungs (31+/-36 at 5 min to 1821+/-83 pmol ml(-1) at 60 min) than in normoxic rat lungs (329+/-20 to 1281+/-127 pmol ml(-1), P<0.05). 3. In isolated lungs preconstricted with U-46619, pretreatment with DMPPO (1 microM) potentiated the vasodilator effects of atrial natriuretic peptide (100 pM-10 nM) and sodium nitroprusside (1 pM 10 nM), but did not alter vasodilation to isoproterenol. 4. In conscious rats previously exposed to 15 days hypoxia and studied under 10% O2, DMPPO (0.01, 0.05 and 0.1 mg kg(-1), i.v. bolus) caused a dose-dependent decrease in pulmonary arterial pressure (Pap) with no change in systemic artery pressure (Sap) and cardiac output. 5. Continuous infusion of DMPPO (0.1 mg kg(-1) h(-1) i.v. by osmotic pumps) in rats exposed to 10% O2 during 2-weeks reduced the Pap (P<0.05) and the degree of muscularization of pulmonary vessels at the alveolar wall (P<0.01) and alveolar duct levels (P<0.05) despite no significant change in right ventricular hypertrophy. 6. These results suggest that cyclic GMP phosphodiesterase inhibition may selectively dilate pulmonary circulation during chronic hypoxia.

Topics: Allopurinol; Animals; Atrial Natriuretic Factor; Cyclic GMP; Drug Interactions; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; In Vitro Techniques; Isoproterenol; Male; Myocardial Contraction; Nitroprusside; Phosphodiesterase Inhibitors; Rats; Rats, Wistar; Time Factors; Vasodilation

Topics: Aging; Animals; Cyclic GMP; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Heart; Hypoxia; In Vitro Techniques; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Rabbits; Shock, Septic; Stress, Physiological; Transcription, Genetic; Ventricular Function, Left; Ventricular Function, Right

Recent reports indicate the presence of two carbon monoxide (CO)-inducing enzymes, heme oxygenase (HO)-1 and -2 in airway smooth muscle. Generally HO-2 is considered to be a constitutive enzyme associated with various neuronal structures, whereas HO-1 can be induced by several factors, including hypoxia. Recent functional data indicate that exogenous CO can induce bronchodilation via a NO-independent, cyclic GMP-related mechanism. The aim of the present study was to investigate the potential role of CO as an endogenously produced airway messenger using an in vivo model of airway hypoxia. HO-1 and HO-2-like immunoreactivities were seen in airway smooth muscle along the bronchus and in the respiratory epithelium. The staining for HO-1 was relatively weak but consistent in all animals investigated. In contrast, the HO-2 staining was intense at all locations. After hypoxic stimulation, the staining for HO-1 and HO-2 was equally intense, indicating an up-regulation of the HO-1 expression. In another set up, anaesthetized, ventilated guinea-pigs were given a continuous infusion of histamine to increase total pulmonary resistance (R1). Hypoxic stimulation, induced by inhalation of 180 breaths of pure nitrogen (N2), resulted in a subsequent reduction in R1. Pretreatment with Rp-8Br-cGMPs, a cyclic GMP antagonist abolished more than 75% of this reduction, whereas L-NAME, an antagonist of NO synthesis, was without effect. Zinc protoporphyrin-IX (ZnPP), an inhibitor of HO, mimicked the effects of Rp-8Br-cGMPS. In conclusion, the present findings suggest a possible role for CO in the hypoxic regulation of airway tone.

Topics: Airway Resistance; Animals; Bronchi; Carbon Monoxide; Cyclic GMP; Disease Models, Animal; Guinea Pigs; Heme Oxygenase (Decyclizing); Hypoxia; Immunohistochemistry; Male; Muscle, Smooth; Trachea

Previously, it had been observed that nitric oxide (NO) contributes to hypoxia-induced pial artery dilation in the newborn pig. Additionally, it was also noted that activation of ATP-sensitive K+ channels (KATP) contribute to cGMP-mediated as well as to hypoxia-induced pial dilation. Although somewhat controversial, adenosine is also thought to contribute to hypoxic cerebrovasodilation. The present study was designed to investigate the role of NO, cyclic nucleotides, and activation of KATP channels in the elicitation of adenosine's vascular response and relate these mechanisms to the contribution of adenosine to hypoxia-induced pial artery dilation. The closed cranial window technique was used to measure pial diameter in newborn pigs. Hypoxia-induced artery dilation was attenuated during moderate (PaO2 approximately 35 mm Hg) and severe hypoxia (PaO2 approximately 25 mm Hg) by the adenosine receptor antagonist 8-phenyltheophylline (8-PT) (10(-5) M) (26 +/- 2 vs. 19 +/- 2 and 34 +/- 2 vs. 22 +/- 2% for moderate and severe hypoxia in the absence vs. presence of 8-PT, respectively). This concentration of 8-PT blocked pial dilation in response to adenosine (8 +/- 2, 16 +/- 2, and 23 +/- 2 vs. 2 +/- 2, 4 +/- 2, and 6 +/- 2% for 10(-8), 10(-6), and 10(-4) M adenosine before and after 8-PT, respectively). Similar data were also obtained using adenosine deaminase as a probe for the role of adenosine in hypoxic pial dilation. Adenosine-induced dilation was associated with increased CSF cGMP concentration (390 +/- 11 and 811 +/- 119 fmol/ml for control and 10(-4) M adenosine, respectively). The NO synthase inhibitor, L-NNA, and the cGMP antagonist, Rp 8-bromo cGMPs, blunted adenosine-induced pial dilation (8 +/- 1, 14 +/- 1, and 20 +/- 3 vs. 3 +/- 1, 5 +/- 1, and 8 +/- 3% for 10(-8), 10(-6), and 10(-4) M adenosine before and after L-NNA, respectively). Adenosine dilation was also blunted by glibenclamide, a KATP antagonist (9 +/- 2, 14 +/- 3, 21 +/- 4 vs. 4 +/- 1, 8 +/- 2, and 11 +/- 2% for 10(-8), 10(-6), and 10(-4) M adenosine before and after glibenclamide, respectively). Finally, it was also observed that adenosine-induced dilation was associated with increased CSF cAMP concentration and the cAMP antagonist, Rp 8-bromo cAMPs, blunted adenosine pial dilation. These data show that adenosine contributes to hypoxic pial dilation. These data also show that NO, cGMP, cAMP, and activation of KATP channels all contribute to adenosine induced pial dilation. Finally, the

Topics: Adenosine; Adenosine Deaminase; Adenosine Triphosphate; Animals; Arteries; Blood Pressure; Cyclic AMP; Cyclic GMP; Enkephalin, Leucine; Enkephalin, Methionine; Female; Glyburide; Hypoxia; Male; Nitric Oxide; Nucleotides, Cyclic; Pia Mater; Potassium Channels; Swine; Theophylline; Vasodilation; Vasodilator Agents

It has been previously observed that nitric oxide (NO) contributes to hypoxic pial artery dilation and that both sodium nitroprusside (SNP), a releaser of NO, and hypoxia elicit dilation via activation of ATP-sensitive K+ channels in the newborn pig. Other studies, however, have shown that NO activates calcium-sensitive K+ (K(Ca)) channels. The present study, therefore, was designed to investigate the role of K(Ca)-channel activation in NO and hypoxic dilation and to relate this mechanism to the previously observed role of NO in hypoxic dilation in newborn pigs equipped with closed cranial windows. SNP (10(-8) and 10(-6) M) elicited pial artery dilation that was unchanged in the presence of the K(Ca)-channel antagonist iberiotoxin (10(-7) M; 10 +/- 1 and 20 +/- 1 vs. 9 +/- 1 and 20 +/- 2% for 10(-8) and 10(-6) M SNP in the absence and presence of iberiotoxin, respectively). Responses to S-nitroso-N-acetylpenicillamine and 8-bromoguanosine 3',5'-cyclic monophosphate were similarly unchanged by iberiotoxin. In contrast, iberiotoxin attenuated the dilation resulting from moderate and severe hypoxia (arterial PO2 approximately 35 and 25 mmHg, respectively; 27 +/- 1 vs. 21 +/- 2 and 34 +/- 1 vs. 16 +/- 2% for moderate and severe hypoxia in the absence and presence of iberiotoxin, respectively). Iberiotoxin blocked responses to the K(Ca)-channel agonist NS-1619, whereas responses to the ATP-sensitive K+ agonist cromakalim were unchanged (8 +/- 1 and 15 +/- 1 vs. 1 +/- 1 and 1 +/- 1% for 10(-8) and 10(-6) M NS-1619 in the absence and presence of iberiotoxin, respectively). These data show that NO and guanosine 3',5'-cyclic monophosphate do not elicit dilation via K(Ca)-channel activation. However, activation of K(Ca) channels does contribute to hypoxic pial dilation. Finally, these data suggest that substances other than NO are involved in the contribution of K(Ca)-channel activation to hypoxic pial artery dilation.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Animals, Newborn; Arterioles; Benzimidazoles; Benzopyrans; Cerebral Arteries; Cromakalim; Cyclic GMP; Female; Hypoxia; Male; Muscle, Smooth, Vascular; Nitric Oxide; Nitroprusside; Penicillamine; Pia Mater; Potassium Channels; Pyrroles; S-Nitroso-N-Acetylpenicillamine; Swine; Vasodilation; Vasodilator Agents

We investigated the effects of chronic hypoxia on the major outward K+ currents in early cultured human main pulmonary arterial smooth muscle cells (HPSMC). Unitary currents were measured from inside-out, outside-out, and cell-attached patches of HPSMC. Chronic hypoxia depolarized resting membrane potential (Em) and reduced the activity of a charybdotoxin (CTX)- and iberiotoxin-sensitive, Ca2+-dependent K+ channel (KCa). The 4-aminopyridine-sensitive and CTX-insensitive channel or the delayed rectifier K+ channel was unaffected by chronic hypoxia. Chronic hypoxia caused a +33- to +53-mV right shift in voltage-dependent activation of K(Ca) and a decrease in K(Ca) activity at all cytosolic Ca2+ concentrations ([Ca2+]i) in the range of 0.1-10 microM. Thus the hypoxia-induced decrease in K(Ca) activity was most likely due to a decrease in K(Ca) sensitivity to Em and [Ca2+]i. Chronic hypoxia reduced the ability of nitric oxide (NO.) and guanosine 3',5'-cyclic monophosphate (cGMP) to activate K(Ca). The cGMP-dependent protein kinase-induced activation of K(Ca) was also significantly inhibited by chronic hypoxia. In addition, inhibiting channel dephosphorylation with calyculin A caused significantly less increase in K(Ca) activity in membrane patches excised from chronically hypoxic HPSMC compared with normoxic controls. This suggests that the mechanism by which hypoxia modulates NO.-induced K(Ca) activation is by decreasing the NO./cGMP-mediated phosphorylation of the channel.

Topics: Blood Vessels; Calcium; Chronic Disease; Cyclic GMP; Electrophysiology; Humans; Hypoxia; Membrane Potentials; Muscle, Smooth, Vascular; Nitric Oxide; Potassium Channels; Protein Kinases; Pulmonary Circulation

1. Studies were performed on rats that had been made chronically hypoxic (CH rats) in a normoxic chamber at 12% O2 for 3-5 weeks. Under Saffan anaesthesia, respiratory and cardiovascular variables, renal haemodynamics and renal function were recorded while the rats spontaneously breathed 12% O2 followed by a switch to air breathing for 20 min. Plasma renin activity was assessed by radioimmunoassay of angiotensin I. Plasma atrial natiruetic peptide (ANP) was indirectly assessed by measurement of cyclic GMP in urine. 2. When breathing 12% O2, CH rats showed hyperventilation and raised haematocrit (52%) relative to normoxic (N) rats. But arterial pressure (ABP), renal blood flow (RBF), renal vascular conductance (RVC), mean right atrial pressure (mRAtP), urine flow, glomerular filtration rate (GFR) and absolute sodium excretion (UNaV) were comparable to those recorded in N rats breathing air. Urinary cGMP was 40% greater than in N rats, but plasma renin activity was not significantly greater in CH than in N rats. 3. Air breathing in CH rats induced hypoventilation, a 12% increase in ABP, no change in mRAtP, RBF or GFR, but increases of 75 and 100% in urine flow and UNaV, respectively. Neither urinary cGMP nor plasma renin activity changed. Such increases in urine flow and UNaV were absent when renal perfusion pressure (RPP) was prevented from rising during air breathing by using an occluder on the dorsal aorta. 4. We propose that by 3-5 weeks of chronic hypoxia renal function was normalized, principally because arterial O2 content was normalized by the increase in haematocrit and because ABP and renal haemodynamics were normalized: acute hypoxia in N rats produces a fall in ABP. We suggest that plasma ANP was raised by the actions of hypoxia or erythropoietin on the atrium, rather than by atrial distension, but suggest that ANP had little direct influence on renal function and tended to limit the influence of the renin-angiotensin system. We further propose that the diuresis and natriuresis seen during air breathing were mediated by the increase in RPP; neither plasma ANP nor renin activity change in the immediate short term.

Topics: Animals; Atrial Natriuretic Factor; Blood Gas Analysis; Cyclic GMP; Hematocrit; Hypoxia; Kidney; Male; Rats; Rats, Wistar; Renin; Renin-Angiotensin System; Respiration

Neonatal pulmonary hypertension is associated with increased pulmonary vascular reactivity. We studied the responses of isolated porcine intrapulmonary arteries after exposure of piglets to chronic hypobaric hypoxia (CHH) from 0 to 2.5, 3 to 6, or 14 to 17 days of age. CHH inhibited the postnatal development of endothelium-dependent vasorelaxation to acetylcholine (ACh) and the calcium ionophore A-23187. Basal accumulation of guanosine 3', 5'-cyclic monophosphate (cGMP) was unaffected, but cGMP response to ACh was inhibited. Endothelium-independent relaxation to nitric oxide (NO) and zaprinast (a phosphodiesterase inhibitor) was also inhibited, but cGMP accumulation in response to these agonists was normal. The ability of sodium nitroprusside (SNP) to cause vasorelaxation and increase cGMP accumulation was unaffected. Contractile responses to potassium chloride and prostaglandin F2 alpha (PGF2 alpha) were similar to normal after exposure from birth and 3 days and were decreased in the older group, but the ability of NG-monomethyl-L-arginine acetate to increase PGF2 alpha-induced contractions decreased. Thus exposure of newborn piglets to CHH causes 1) no increase in contractile responses and 2) impairment of endothelium-dependent and -independent relaxation by impairing signal transduction mechanisms involved in the release of NO and the effectiveness of cGMP.

Topics: Acetylcholine; Animals; Animals, Newborn; Atmospheric Pressure; Calcimycin; Chronic Disease; Cyclic GMP; Dinoprost; Endothelium, Vascular; Heart; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Nitric Oxide; omega-N-Methylarginine; Potassium Chloride; Pulmonary Artery; Purinones; Swine; Vasodilation; Vasomotor System

Perfusate levels of nitric oxide (NO)-containing compounds and guanosine 3',5'-cyclic monophosphate (cGMP) are increased in hypoxia-induced hypertensive rat lungs. To test if increased cGMP was due to NO stimulation of soluble guanylate cyclase (sGC), we examined effects of inhibition of NO synthase with N omega-nitro-L-arginine (L-NNA) on perfusate accumulation of cGMP in physiological salt solution (PSS)-perfused hypertensive lungs isolated from rats exposed for 3-4 wk to hypobaric hypoxia. Because 200 microM L-NNA did not reduce cGMP, we next examined inhibitors of other pathways of stimulation of either sGC or particulate GC (pGC). Neither 5 microM Zn-protophorphyrin, an inhibitor of CO production by heme oxygenase, nor 10 mM aminotriazole, an inhibitor of H2O2 metabolism by catalase, reduced perfusate cGMP. However, an antiserum to atrial natriuretic peptide (ANP; 100 microliters antiserum/30 ml PSS), to inhibit ANP activation of pGC, completely prevented accumulation of the nucleotide. ANP antiserum was also more effective than L-NNA in reducing lung tissue cGMP. In contrast, L-NNA but not ANP antiserum increased resting vascular tone. These results suggested that whereas ANP determined perfusate and tissue levels of cGMP, NO regulated vascular tone. To test if perfusate cGMP reflected ANP stimulation of pGC in endothelial rather than smooth muscle cells, we examined effects of 10 microM Zaprinast, an inhibitor of cGMP hydrolysis in smooth muscle but not endothelial cells, and found no increase of cGMP in hypertensive lungs. ANP levels were not elevated in hypertensive lungs, and it is unclear by what mechanism the ANP-stimulated activity of pGC is increased in hypertensive pulmonary vascular endothelial cells.

Topics: Altitude; Amitrole; Animals; Atrial Natriuretic Factor; Catalase; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; Heme Oxygenase (Decyclizing); Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Immune Sera; Kinetics; Lung; Male; Nitroarginine; Protoporphyrins; Purinones; Rats; Rats, Sprague-Dawley; Reference Values

Hypoxia activates erythropoietin-producing cells, chemoreceptor cells of the carotid body and pulmonary artery smooth muscle cells (PSMC) with a comparable arterial PO2 threshold of some 70 mmHg. The inhibition by CO of the hypoxic responses in the two former cell types has led to the proposal that a haemoprotein is involved in the detection of the PO2 levels. Here, we report the effect of CO on the hypoxic pulmonary vasoconstriction (HPV). Pulmonary arterial pressure (PAP) was measured in an in situ, blood-perfused lung preparation. PAP in normoxia (20% O2, 5% CO2) was 15.2+/-1.8 mmHg, and hypoxia (2% O2, 5% CO2) produced a DeltaPAP of 6.3+/-0.4 mmHg. Addition of 8% or 15% CO to the hypoxic gas mixture reduced the DeltaPAP by 88.3+/-2.7% and 78.2+/-6.1% respectively. The same levels of CO did not affect normoxic PAP nor reduced the DeltaPAP produced by angiotensin II. The effect of CO was studied after inhibition of the NO-cyclic guanosine monophosphate (cGMP) cascade with N-methyl-l-arginine (5.10(-5) M) or methylene blue (1.4.10(-4) M). It was found that both inhibitors more than doubled the hypoxic DeltaPAP without altering the effectiveness of CO to inhibit the HPV. In in vitro experiments we verified the inhibition of guanylate cyclase by measuring the levels of cGMP in segments of the pulmonary artery. Cyclic GMP levels were 1.4+/-0.2 (normoxia), 2.5+/-0.3 (hypoxia) and 3.3+/-0.5 pmole/mg tissue (hypoxia plus 8% CO); sodium nitroprusside increased normoxic cGMP levels about fourfold. Methylene blue reduced cGMP levels to less than 10% in all cases, and abolished the differences among normoxic, hypoxic and hypoxic plus CO groups. It is concluded that CO inhibits HPV by a NO-cGMP independent mechanism and it is proposed that a haemoprotein could be involved in O2-sensing in PSMC.

Topics: Animals; Carbon Monoxide; Cyclic GMP; Enzyme Inhibitors; Female; Guanylate Cyclase; Hypoxia; In Vitro Techniques; Methylene Blue; Nitric Oxide Synthase; Nitroarginine; Pulmonary Artery; Pulmonary Circulation; Rats; Rats, Wistar; Solubility; Vasoconstriction

To examine the contribution of the cyclic guanosine monophosphate (cGMP) pathway in changes in pulmonary vasoconstriction during the initial days of altitude exposure, we tested the effects of LY83583 (an inhibitor of guanylate cyclase activation) and those of N(G)-monomethyl-L-arginine (an inhibitor of nitric oxide synthesis) on airway hypoxia- (3% O2) and angiotensin II- (AII, 0.2 microg) induced vasoconstrictions in lungs from the rats exposed to either moderate altitude (MA, 570 torr) or high altitude (HA, 430 torr) At 2 days' exposure, hypoxic response was significantly blunted compared with the response in low-altitude (LA, 710 torr) lungs in an altitude-dependent manner. At 7 days' exposure, the response was recovered fully in MA lungs but partially in HA lungs. AII response was not significantly blunted at 2 days' exposure, but was significantly augmented in an altitude-dependent manner at 7 days' exposure. LY83583 (10 micromol L(-1)) potentiated both responses in LA lungs but did not significantly potentiate either response in any altitude-exposed lungs. N(G)-monomethyl-L-arginine (10 micromol L(-1)) potentiated both responses in LA lungs but did not significantly potentiate either response in HA lungs at 2 days' and 7 days' exposure. Thus the cGMP pathway is not responsible for either the change in hypoxic vasoconstriction or the change in AII vasoconstriction in rat lungs during the initial 7 days of altitude exposure.

Topics: Altitude; Animals; Cyclic GMP; Enzyme Inhibitors; Guanylate Cyclase; Hypertrophy, Left Ventricular; Hypertrophy, Right Ventricular; Hypoxia; In Vitro Techniques; Male; Nitric Oxide; Nitric Oxide Synthase; Perfusion; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Vasoconstriction; Vasoconstrictor Agents

Nitric oxide (NO) contributes to hypoxia-induced pial artery dilation, at least in part, via the formation of guanosine 3',5'-cyclic monophosphate (cGMP) and subsequent release of Met-enkephalin and Leu-enkephalin in the newborn pig. In separate studies, these opioids were also observed to elicit NO-dependent pial dilation. The present study was designed to investigate the role of the neuronal isoform of NO synthase (NOS) in hypoxic pial dilation, associated opioid release, and opioid dilation in piglets equipped with a closed cranial window. Tetrodotoxin (10(-6) M) attenuated the dilation resulting from hypoxia (PO2 approximately 35 mmHg; 25 +/- 1 vs. 14 +/- 1%). Similarly, 7-nitroindazole, sodium salt (7-NINA, 10(-6) M), a purported neuronal NOS inhibitor, attenuated hypoxic pial dilation (26 +/- 1 vs. 14 +/- 2%). Hypoxic dilation was accompanied by elevated cerebrospinal (CSF) cGMP, which was blocked by 7-NINA (433 +/- 19 and 983 +/- 36 vs. 432 +/- 19 and 441 +/- 19 fmol/ml for control and hypoxia in absence and presence of 7-NINA, respectively). Additionally, hypoxic dilation was also accompanied by elevated CSF Met-enkephalin, which was attenuated by 7-NINA (1,027 +/- 47 and 2,871 +/- 134 vs. 779 +/- 78 and 1,551 +/- 42 pg/ml for control and hypoxia in absence and presence of 7-NINA, respectively). In contrast, Met-enkephalin (10(-10), 10(-8), and 10(-6) M) induced dilation that was unchanged by 7-NINA (7 +/- 1, 12 +/- 1, and 18 +/- 1 vs. 6 +/- 1, 10 +/- 1, and 17 +/- 1%, respectively). N-methyl-D-aspartate (NMDA, 10(-8) and 10(-6) M), an activator of neuronal NOS, induced pial dilation that was blocked by 7-NINA (10 +/- 1 and 20 +/- 2 vs. 1 +/- 1 and 2 +/- 1%, respectively). However, sodium nitroprusside-induced dilation was unchanged by 7-NINA. These data indicate that neuronal NOS contributes to hypoxic pial artery dilation but not to opioid-induced dilation. Furthermore, these data suggest that neuronally derived NO contributes to hypoxic dilation, at least in part, via formation of cGMP and the subsequent release of opioids.

Topics: Animals; Cerebral Arteries; Cyclic GMP; Endorphins; Enkephalins; Enzyme Inhibitors; Female; Hypoxia; Indazoles; Male; Neurons; Nitric Oxide; Nitric Oxide Synthase; Pia Mater; Swine; Tetrodotoxin; Vasodilation

While it is known that nitric oxide (NO) is an important modulator of tone in the hypertensive pulmonary circulation, the roles of cyclic 3'-5'-guanosine monophosphate (cGMP) and cGMP-phosphodiesterase (PDE) are uncertain. We found that isolated lung perfusate levels of cGMP were over ninefold elevated in hypertensive vs. normotensive control rats. 98-100% of lung cGMP hydrolytic activity was cGMP-specific PDE5, with no significant decrease in PDE activity in hypertensive lungs, suggesting that the elevation in cGMP was due to accelerated production rather than reduced degradation. In pulmonary hypertensive rat lungs, in vitro, cGMP-PDE inhibition by E4021[1-(6-chloro-4-(3,4-methylbenzyl) amino-quinazolin-2-yl)piperdine-4-carboxylate], increased perfusate cGMP threefold, reduced hypoxic vasoconstriction by 58 +/- 2%, and reduced baseline pulmonary artery pressure by 37 +/- 5%. In conscious, pulmonary hypertensive rats, intravenous administration of E4021 reduced hypoxic vasoconstriction by 68 +/- 8%, pulmonary artery pressure by 12.6 +/- 3.7% and total pulmonary resistance by 13.1 +/- 6.4%, with no significant effect on cardiac output, systemic pressure, and resistance. Comparison of E4021 to inhaled nitric oxide demonstrated that cGMP-PDE inhibition was as selective and as effective as inhaled NO.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Calcium Channel Blockers; Cyclic GMP; Diltiazem; Hemodynamics; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Lung; Male; Nitric Oxide; Perfusion; Phosphodiesterase Inhibitors; Piperidines; Pulmonary Circulation; Purinones; Quinazolines; Rats; Rats, Sprague-Dawley; Vasodilation

To elucidate the effect of hypoxia on nitrovasodilator-induced pulmonary vasodilation, we studied canine pulmonary arterial rings under isometric conditions in vitro. Exposure to hypoxia inhibited the relaxant responses of KCl-contracted tissues to sodium nitroprusside (SNP), so that the maximal relaxation (Emax) and the negative logarithm of molar concentration required to produce 50% relaxation (pD2) were decreased from 92 +/- 7 to 62 +/- 5% and from 5.8 +/- 0.2 to 4.7 +/- 0.3, respectively (means +/- SE, P < 0.01 for each). This effect was likewise observed when 8-bromoguanosine-3',5'-cyclic monophosphate was used as a relaxant. The impairment of SNP-induced relaxation of endothelium-denuded rings under hypoxia was abolished by ouabain or K(+)-free solution. Incubation with SNPincreased intracellular cGMP contents in a dose dependent manner, an effect that was not altered by hypoxia. SNP also increased ouabain-sensitive 86Rb uptake, and this effect was inhibited by hypoxia. These results suggest that hypoxia reduces nitrovasodilator-induced relaxation of pulmonary artery, probably through an inhibition of cGMP-dependent sarcolemmal Na-K-adenosine triphosphatase activity.

Topics: Animals; Cyclic GMP; Dogs; Female; Hypoxia; Male; Nitroprusside; Pulmonary Artery; Sodium-Potassium-Exchanging ATPase; Vasodilation

It has previously been observed that nitric oxide (NO) and the opioids Met- and Leu-enkephalin contribute to hypoxia-induced pial artery dilation in the newborn pig. The present study was designed to investigate the relationship between NO and opioids in hypoxic pial dilation. Piglets equipped with closed cranial windows were used to measure pial artery diameter and collect cortical periarachnoid cerebrospinal fluid (CSF) for assay of opioids. Sodium nitroprusside (SNP; 10(-8) and 10(-6) M) elicited pial dilation that was blunted by the soluble guanylate cyclase inhibitor LY-83583 (10(-5) M; 10 +/- 1 and 23 +/- 1 vs. 3 +/- 1 and 7 +/- 1% for 10(-8) and 10(-6) M SNP before and after LY-83583, respectively). SNP-induced dilation was accompanied by increased CSF Met-enkephalin, and coadministration of LY-83583 with SNP blocked these increases in CSF opioid concentration (1,144 +/- 59, 2,215 +/- 165, and 3,413 +/- 168 vs. 1,023 +/- 16, 1,040 +/- 18, and 1,059 +/- 29 pg/ml for control and 10(-8) and 10(-6) M SNP before and after LY-83583, respectively). SNP-induced release of CSF Leuenkephalin was also blocked by LY-83583. Similar blunted vascular and biochemical effects of SNP were observed with coadministration of the purported guanosine 3', 5'-cyclic monophosphate (cGMP) antagonist, the phosphorothioate analogue of 8-bromo-cGMP (BrcGMP) [(R)-p-BrcGMP[S]; 10(-5) M]. The cGMP analogue, BrcGMP, elicited dilation that was also accompanied by increased CSF Met- and Leu-enkephalin. Vascular and biochemical effects of BrcGMP were blunted by (R)-p-cGMP[S] and unchanged by LY-83583. Hypoxia-induced pial artery dilation was attenuated by N omega-nitro-L-arginine (L-NNA; 10(-6) M), an NO synthase inhibitor (25 +/- 2 vs. 14 +/- 1%). Hypoxic pial dilation was accompanied by increased CSF Met-enkephalin, and these increases were attenuated by L-NNA (1,137 +/- 60 and 3,491 +/- 133 vs. 927 +/- 25 and 2,052 +/- 160 pg/ml for control and hypoxia before and after L-NNA, respectively). Hypoxia also increased CSF Leuenkephalin, and these CSF changes were similarly attenuated by L-NNA. These data show that cGMP increases CSF Met- and Leu-enkephalin. Furthermore, these data suggest that NO contributes to hypoxic dilation, at least in part, via formation of cGMP and the subsequent release of opioids.

Topics: Aminoquinolines; Animals; Cerebral Arteries; Cyclic GMP; Enzyme Inhibitors; Female; Hypoxia; Male; Narcotics; Nitric Oxide; Nitroprusside; Swine; Vasodilation

Topics: Animals; Argininosuccinic Acid; Cyclic GMP; Hypercapnia; Hypoxia; In Vitro Techniques; Male; Nitric Oxide; Perfusion; Pulmonary Circulation; Rabbits; Vasoconstriction

Almost all of the studies evaluating the effect of chronic hypoxia on lung nitric oxide production have been performed in adult animals. Because results of studies in adult lungs should not be extrapolated to represent the newborn lung, we performed studies to determine whether decreased nitric oxide production might be involved in the pathogenesis of chronic hypoxia-induced pulmonary hypertension in newborns. We kept newborn pigs in chambers filled with room air (control) or 11-12% O2 for either 3-5 (short) or 10-12 (long) days. Using isolated lungs, we measured pulmonary vascular responses to agents that either stimulate or inhibit the synthesis of nitric oxide. To define the vascular sites of altered production of nitric oxide, we applied the micropuncture technique and measured small venular pressures before and after treatment with a nitric oxide synthesis inhibitor. Pulmonary vascular responses to acetylcholine were blunted in chronically hypoxic piglets of both the short and long groups. The nitric oxide synthesis inhibitor had a different effect in the lungs of control piglets than in those of chronically hypoxic piglets of the long but not of the short group. For the long group, the nitric oxide synthesis inhibitors caused constriction of both arteries and veins in lungs of control but not of chronically hypoxic piglets. These findings support the idea that decreased pulmonary vascular nitric oxide production occurs with chronic hypoxia in newborn pigs and might therefore contribute to the pathogenesis of pulmonary hypertension in newborns.

Topics: Acetylcholine; Animals; Animals, Newborn; Blood Pressure; Chronic Disease; Cyclic GMP; Endothelium, Vascular; Enzyme Inhibitors; Hypoxia; In Vitro Techniques; Lung; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Pulmonary Circulation; Swine; Vascular Resistance; Vasodilator Agents

Topics: Animals; Carotid Body; Cell Hypoxia; Cells, Cultured; Cyclic AMP; Cyclic GMP; Hypoxia; Ion Transport; Nerve Tissue Proteins; Nitric Oxide; Nucleotides, Cyclic; Patch-Clamp Techniques; Penicillamine; Potassium; Potassium Channels; Rats; S-Nitroso-N-Acetylpenicillamine; Signal Transduction

The present study was designed to investigate the contribution of opioids and nitric oxide (NO) to hypoxia-induced pial vasodilation. Newborn pigs equipped with a closed cranial window were used to measure pial arteriolar diameter and to collect cortical periarachnoid cerebrospinal fluid (CSF) for assay of opioids and guanosine 3',5'-cyclic monophosphate (cGMP). Hypoxia-induced pial dilation was potentiated by norbinaltorphimine, 10(-6) M, a kappa-opioid antagonist (25 +/- 2 vs. 33 +/- 3%, n = 5), but was blunted by beta-funaltrexamine, 10(-8) M, a mu-opioid antagonist (28 +/- 2 vs. 19 +/- 1%, n = 5). Hypoxia-induced vasodilation was associated with increased CSF methionine enkephalin, a mu-opioid agonist (884 +/- 29 vs. 2,638 +/- 387 pg/ml, n = 5). N omega-nitro-L-arginine (L-NNA), an NO synthase inhibitor (10(-6) M), also blunted hypoxia-induced vasodilation that was further diminished by coadministration of L-NNA and beta-funaltrexamine (26 +/- 2, 14 +/- 1, and 9 +/- 1%, respectively, n = 5). Reversal of the above order of antagonist administration resulted in similar inhibition of hypoxia-induced pial dilation. Hypoxia-induced vasodilation was also associated with an increase in CSF cGMP that was attenuated by L-NNA (2.1 +/- 0.1- vs. 1.1 +/- 0.2-fold change in CSF cGMP, n = 5). Sodium nitroprusside (10(-6) M) increased CSF cGMP and methionine enkephalin concentration similar to hypoxia. These data suggest that hypoxia-induced pial arterial vasodilation, in part, is due to NO and/or cGMP-induced methionine enkephalin release as well as the direct action of NO.

Topics: Amino Acid Oxidoreductases; Analysis of Variance; Animals; Animals, Newborn; Arginine; Arterioles; Cerebral Arteries; Cyclic GMP; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Methionine; Enkephalins; Female; Hypoxia; Male; Muscle, Smooth, Vascular; Naltrexone; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Nitroprusside; Receptors, Opioid, kappa; Receptors, Opioid, mu; Swine; Vasodilation

Topics: Amino Acid Oxidoreductases; Animals; Cyclic GMP; Endothelium, Vascular; Humans; Hypertension, Pulmonary; Hypoxia; NADPH Dehydrogenase; Nitric Oxide; Nitric Oxide Synthase; Pulmonary Artery

Numerous studies have demonstrated that carotid sinus nerve fibers mediate a so-called "efferent" inhibition of carotid body chemoreceptors. However, the mechanism(s) underlying this phenomenon are not understood. Recently, it has been shown that an extensive plexus of nitric oxide synthase-containing carotid sinus nerve fibers innervate the carotid body, and that many fine, beaded fibers can be seen in close proximity to small blood vessels as well as lobules of parenchymal cells. The present study examined the effects of centrifugal neural activity in the carotid sinus nerve on the accumulation of [3H]citrulline synthesized from [3H]arginine in the cat carotid body, and the possible involvement of nitric oxide in mediating "efferent" chemoreceptor inhibition. Electrical stimulation of carotid sinus nerve C-fibers evoked an increase in [3H]citrulline accumulation in the carotid body, which was Ca(2+)-dependent and blocked by L-NG-nitroarginine methylester (0.1 mM), an inhibitor of nitric oxide synthase. Using a vascularly perfused in vitro carotid body preparation, chemoreceptor activity was recorded from thin nerve filaments split-off from the main trunk of the carotid sinus nerve. Electrical stimulation of the main nerve trunk at C-fiber intensities inhibited steady-state chemoreceptor discharge, and this effect was blocked by L-NG-nitroarginine methylester. However, when the organ preparation was switched to the superfuse-only mode, carotid sinus nerve stimulation failed to alter the steady-state discharge, but under these conditions, prolonged nerve stimulation (> 5 min) did attenuate the chemoreceptor response to hypoxia, an effect which was likewise blocked by L-NG-nitroarginine methylester. The present data, together with previous anatomical findings that nitric oxide synthase immunoreactivity is present in both sensory and autonomic ganglion cells innervating the carotid body, suggest that two neural mechanisms may be involved in the inhibitory neural regulation of carotid chemoreceptors. One mechanism appears to involve nitric oxide release from intralobular sensory C-fibers, which lie in close proximity to the chemoreceptor type I cells. The other mechanism involves release of nitric oxide from perivascular terminals of autonomic microganglia neurons, which control carotid body blood flow.

Topics: Animals; Arginine; Carotid Body; Cats; Chemoreceptor Cells; Citrulline; Cyclic GMP; Electric Stimulation; Evoked Potentials; Hypoxia; Immunohistochemistry; Nitric Oxide

Carbon monoxide (CO) is a product of the enzyme heme oxygenase (HO; EC 1.14.99.3). In vascular smooth muscle cells, exogenously administered CO increases cyclic guanosine 3',5'-monophosphate (cGMP), which is an important regulator of vessel tone. We report here that smooth muscle cells produce CO via HO and that it regulates cGMP levels in these cells. Hypoxia, which has profound effects on vessel tone, significantly increased the transcriptional rate of the HO-1 gene resulting in corresponding increases of its mRNA and HO enzymatic activity. In addition, under the same conditions, rat aortic and pulmonary artery smooth muscle cells accumulated high levels of cGMP following a similar time course to that of HO-1 production. The increased accumulation of cGMP in smooth muscle cells required the enzymatic activity of HO, since it was abolished by a specific HO inhibitor, tin protoporphyrin. In contrast, N omega-nitro-L-arginine, a potent inhibitor of nitric oxide (NO) synthesis, had no effect on cGMP produced by smooth muscle cells, indicating that NO is not responsible for the activation of guanylyl cyclase in this setting. Furthermore, conditioned medium from hypoxic smooth muscle cells stimulated cGMP production in recipient cells and this stimulation was completely inhibited by tin protoporphyrin or hemoglobin, an inhibitor of CO production and a scavenger of CO, respectively. This report shows that HO-1 is expressed by vascular smooth muscle cells and that its product, CO, may regulate vascular tone under physiologic and pathophysiologic (such as hypoxic) conditions.

Topics: Animals; Base Sequence; Carbon Monoxide; Carboxyhemoglobin; Cells, Cultured; Cyclic GMP; DNA Primers; Gene Expression Regulation, Enzymologic; Heme Oxygenase (Decyclizing); Hypoxia; In Vitro Techniques; Male; Molecular Sequence Data; Muscle, Smooth, Vascular; Rats; Rats, Sprague-Dawley; Transcription, Genetic

Adult respiratory distress syndrome is characterized by hypoxia and acute pulmonary hypertension. Therefore we examined the effect of acute hypoxia on the mechanisms of pulmonary vasodilation.. Isolated rat pulmonary artery rings were suspended on tensiometers in a balanced salt solution. A normoxic gas mixture was bubbled through the solution (21% O2, 5% CO2, 74% N2). Rings were preconstricted with phenylephrine, and the following mechanisms of pulmonary vascular smooth muscle relaxation were studied in a random order: (1) endothelial-dependent cyclic guanosine monophosphate-mediated (acetylcholine, 10(-9) to 10(-6) mol/L), (2) endothelial-independent cyclic guanosine monophosphate-mediated (nitroprusside, 10(-9) to 10(-6) mol/L), and (3) beta-adrenergic receptor cyclic adenine monophosphate-mediated (isoproterenol, 10(-9) to 10(-6) mol/L). Separate rings were preconstricted with phenylephrine, and the gas was switched to a hypoxic mixture (0% O2, 5% CO2, 95% N2). After vasoconstriction to hypoxia reached a plateau, the response to the maximal effective dose of the above vasodilators (10(-6) mol/L) was determined in a random order. Statistical analysis was done with one-way analysis of variance with post hoc Bonferroni-Dunn correction. A p value of less than 0.05 was accepted as significant.. Endothelial-dependent and -independent cyclic guanosine monophosphate-mediated relaxation was the same in normoxia and hypoxia. On the other hand, hypoxia inhibited beta-adrenergic receptor cyclic adenine monophosphate-mediated pulmonary vasorelaxation (97.5% +/- 2.5% versus 71.5% +/- 2.3% in hypoxia; p < 0.01).. These data suggest that hypoxia selectively inhibits beta-adrenergic cyclic adenine monophosphate-mediated pulmonary vasorelaxation. This dysfunction of the normal mechanism of pulmonary vasodilation may contribute to the pulmonary hypertension seen in adult respiratory distress syndrome.

Topics: Acetylcholine; Acute Disease; Animals; Cyclic AMP; Cyclic GMP; Endothelium, Vascular; Hypoxia; In Vitro Techniques; Isoproterenol; Lung; Muscle, Smooth, Vascular; Nitroprusside; Rats; Rats, Sprague-Dawley; Vasodilation

We examined the effects of exercise training on pulmonary arterial blood pressure (Ppa) and on adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (GMP) concentrations in lung tissue at rest and during exercise under hypoxic conditions in catheter-implanted rats. Male Wistar rats were divided into an exercise-trained group (ET, n = 32) and nonexercised control group (control, n = 32). ET rats exercised 40 min/day 6 days/week for 6 weeks, at an altitude of 610 m on a treadmill. The mean Ppa levels of the ET were significantly lower than those of controls at rest and during exercise at 610- and 2500-m altitudes. The exercise-induced mean Ppa increase in the ET was less than that in controls at both 610- and 2500-m altitudes. Resting lung cAMP increased more in the ET than in controls at both 610- and 2500-m altitudes. In ET, cGMP was significantly greater at the 2500-m altitude than at the 610-m altitude at rest and just after exercise. Hypoxic exercise in ET was accompanied by a preferential increase in cGMP but not in cAMP. These results suggest that the intracellular augmentation of cAMP and cGMP in ET plays an important role in attenuating hypoxic pulmonary vasoconstriction (HPV) and exercise-induced increases in Ppa.

Topics: Animals; Blood Pressure; Cyclic AMP; Cyclic GMP; Heart Rate; Hypoxia; Lung; Male; Oxygen; Physical Conditioning, Animal; Pulmonary Circulation; Rats; Rats, Wistar; Vasoconstriction

It is believed that hypoxia results in the release of neurotransmitters in the central nervous system, which can excite or inhibit breathing. Recent evidence indicates that nitric oxide (NO) is a physiological messenger molecule that may serve as a neurotransmitter in the CNS. In this study we examined (1) the localization of nitric oxide synthase (NOS) within the nucleus tractus solitarius, and (2) the role of the NO-cGMP pathway in the respiratory response to oxygen deprivation. Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry was used to determine the distribution of neurons that express NOS, an enzyme involved in NO formation. The NOS inhibitor N omega-nitro-L-arginine was used as tool to assess the NOS activity in the medulla, and to define the role of NO in the respiratory response to acute oxygen deprivation. In the rat and the cat brainstem, histochemical studies showed the presence of NADPH-diaphorase reactive neurons within subnuclei of the nucleus tractus solitarius which receive peripheral chemoreceptor inputs. Chronic pretreatment of rats with N omega-nitro-L-arginine (75 mg/kg, ip, twice daily for 7 days) caused a significant decrease in cGMP, and attenuated the ventilatory response to hypoxia. In anesthetized, paralyzed, vagotomized and artificially ventilated cats with intact carotid sinus nerves (n = 8), administration of N omega-nitro-L-arginine (30-100 mg/kg) attenuated the response to hypoxia, and caused the hypoxia induced roll-off of phrenic nerve activity to occur significantly earlier than when NOS activity was not inhibited. In sinoaortic denervated cats (n=9) blockage of NOS potentiated the decline of the phrenic nerve output. The data suggest that oxygen deprivation leads to activation of NO-cGMP pathway in the central nervous system, which contributes to the induction and maintenance of hypoxia-induced increase in respiratory output. In addition, these findings indicate that NO may inhibit inhibitory synaptic transmission that is triggered by CNS hypoxia, and this is not directly related to peripheral chemoreceptor inputs.

Topics: Animals; Arginine; Blood Pressure; Carotid Body; Cats; Cyclic GMP; Enzyme Inhibitors; Heart Rate; Hypoxia; Medulla Oblongata; NADPH Dehydrogenase; Neurons; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Oxygen; Partial Pressure; Rats; Rats, Sprague-Dawley; Respiration; Solitary Nucleus

Working rat hearts perfused with 5.5 mM glucose were submitted to a 10-min period of no-flow ischaemia or anoxia. Both conditions stimulated glycogenolysis, activated phosphorylase and increased cyclic GMP content, although the time course of these changes differed in anoxia and ischaemia. Changes in cyclic GMP content were not correlated with glycogenolysis or phosphorylase activation. Perfusion with 1 microM L-nitroarginine methylester, an inhibitor of nitric oxide synthase, decreased cGMP concentration under normoxic conditions and abolished the ischaemia-induced increase in cGMP. The inhibitor decreased the coronary flow without affecting the overall working performance of the hearts under normoxic conditions.

Topics: Adenosine Triphosphate; Amino Acid Oxidoreductases; Animals; Arginine; Coronary Circulation; Cyclic GMP; Fructosediphosphates; Glucose; Glycogen; Heart; Hexosephosphates; Hypoxia; In Vitro Techniques; Kinetics; Male; Myocardial Ischemia; Myocardium; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Perfusion; Rats; Rats, Wistar; Time Factors

Myocardial hypoxia is known to be accompanied by the release of atrial natriuretic factor (ANF), a peptide which dilates the coronary vessels by stimulating particulate guanylyl cyclase. We have assessed whether ANF plays a paracrine role in hypoxic coronary vasodilatation, a reaction which we had previously found to be associated with increased cyclic GMP production. Compound HS 142-1 (100 micrograms/ml), a specific antagonist of the guanylyl cyclase ANF receptor, inhibited by 50-70% the coronary-vasodilating effects of human ANF (1-10 micrograms) administered to isolated guinea pig hearts, but affected neither hypoxic coronary vasodilation nor cyclic GMP overflow. In contrast, the nitric oxide synthase inhibitor N omega-methyl-L-arginine (300 microM) reduced hypoxic coronary vasodilatation and cyclic GMP overproduction by approximately 70% and 50-60%, respectively. Thus, unlike nitric oxide, ANF appears not to play a paracrine role in hypoxic coronary vasodilatation.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Atrial Natriuretic Factor; Coronary Circulation; Cyclic GMP; Guanylate Cyclase; Guinea Pigs; Humans; Hypoxia; In Vitro Techniques; Male; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Perfusion; Polysaccharides; Receptors, Atrial Natriuretic Factor; Vasodilation

To evaluate the potency of vasodilatory drugs in hypoxia, we studied the effects of nitroglycerin (NTG), prostaglandin E1 (PGE1), and hydralazine on porcine coronary artery constricted with endothelin-1 (ET-1) in both oxygenated and hypoxic conditions. Removal of endothelium potentiated NTG-induced relaxation in oxygenated conditions. Hypoxia potentiated relaxation of endothelium-intact arteries induced by NTG, but not relaxation of endothelium-denuded arteries. These findings suggest that hypoxia may modify endothelial function in NTG-induced relaxation. The relaxation of endothelium-intact and -denuded arteries induced by PGE1 in hypoxia was significantly greater than that in the oxygenated condition. PGE1 significantly increased the content of cyclic AMP in the hypoxic condition; it was much greater than that in the oxygenated condition, suggesting that hypoxia may enhance PGE1-induced relaxation by increasing cyclic AMP levels. Hypoxia attenuated hydralazine-induced relaxation in both endothelium-intact and denuded arteries. Indomethacin and aspirin attenuated hydralazine-induced relaxation in the oxygenated condition, suggesting that cyclooxygenase-related eicosanoid(s) may be involved in hydralazine-induced relaxation. However, indomethacin did not alter relaxation of hypoxic arteries induced by hydralazine. These findings suggest that hypoxia may inactivate cyclooxygenase in hydralazine-induced relaxation. Hypoxia may greatly modify the action of vasodilators on porcine coronary smooth muscle.

Topics: Alprostadil; Analysis of Variance; Animals; Coronary Vessels; Cyclic AMP; Cyclic GMP; Endothelins; Hydralazine; Hypoxia; In Vitro Techniques; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroglycerin; Swine

Acute hypoxia causes pulmonary hypertension in the fetus and newborn that is contrasted by systemic hypotension or normotension. To better understand the role of nitric oxide (NO) in this specific pulmonary vascular response, we determined the acute effects of decreased oxygenation on NO production in ovine fetal pulmonary and systemic (mesenteric) endothelial cells. NO was assessed by measuring cGMP accumulation in fetal vascular smooth muscle (VSM) cells during co-culture incubations of endothelium and VSM (40 s) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Changes in cGMP were dependent on the endothelium and on NO synthase and guanylate cyclase activity. At high O2 (680 mm Hg), basal NO was detectable and NO increased 6- to 10-fold with bradykinin or A23187. In pulmonary endothelium, basal NO fell 58% at pO2 = 150 mm Hg and 51% at 40 mm Hg versus 680 mm Hg, while NO with bradykinin fell 56% and 63%, respectively. NO with A23187, however, was unchanged at 150 mm Hg, but it fell 56% at 40 mm Hg. In contrast, in systemic endothelium basal and stimulated NO production were not altered at lower O2. Findings were similar using pulmonary or systemic detector VSM cells, and exogenous L-arginine had no effect. Thus, decreased O2 acutely attenuates NO production specifically in fetal pulmonary endothelial cells. This process is not related to changes in O2 or L-arginine availability as substrates for NO synthase; alternatively, it may be partially mediated by specific effects of O2 on pulmonary endothelial cell calcium homeostasis.

Topics: 1-Methyl-3-isobutylxanthine; Amino Acid Oxidoreductases; Animals; Bradykinin; Calcimycin; Cells, Cultured; Cyclic GMP; Disease Models, Animal; Endothelium, Vascular; Female; Guanylate Cyclase; Humans; Hypoxia; Infant, Newborn; Lung; Mesenteric Arteries; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Oxygen; Persistent Fetal Circulation Syndrome; Pregnancy; Pulmonary Artery; Sheep

We describe the hemodynamic effects and metabolic fate of inhaled NO gas in 12 anesthetized piglets. Pulmonary and systemic hemodynamic responses to incremental [NO] (5-80 ppm) were tested during ventilation with high- [0.30 inspired O2 fraction (FIO2)] and low-O2 (0.10 FIO2) mixtures. In six animals, inhalation of 40 ppm NO was maintained over 6 h to test effects of prolonged exposure (0.30 FIO2). In the other six animals, pulmonary hypertension was induced by hypoxic ventilation (0.10 FIO2) and responses to NO were tested. Inhaled low [NO] partially reversed pulmonary hypertension induced by alveolar hypoxia; mean pulmonary arterial pressure decreased from 31.4 +/- 2.3 mmHg during hypoxia to 18.2 +/- 1.2 mmHg during 5 ppm NO. Mean pulmonary arterial pressure at 0.10 FIO2 did not fall further at higher [NO] (10-40 ppm) and never reached control levels. Pulmonary vascular resistance increased with institution of hypoxic ventilation and fell with subsequent administration of NO, ultimately reaching control levels. Inhaled NO did not affect systemic vascular resistance. Plasma levels of NO2- + NO3- and methemoglobin (MetHb) levels increased with increasing [NO]. Over 6 h of NO administration during high-O2 ventilation, MetHb equilibrated at subtoxic levels while NO2- + NO3- increased. Nitrosylhemoglobin, analyzed by electron paramagnetic resonance spectrophotometry was not detected in blood at any time. At the relatively low concentrations (5-80 ppm) that are effective in relieving experimental pulmonary hypertension induced by alveolar hypoxia, inhaled NO gas causes accumulation of NO2- + NO3- in plasma and a small increase in MetHb but no detectable nitrosylhemoglobin.

Topics: Administration, Inhalation; Animals; Blood Gas Analysis; Blood Pressure; Cyclic GMP; Electron Spin Resonance Spectroscopy; Hemodynamics; Hemoglobins; Hypoxia; Methemoglobin; Nitric Oxide; Pulmonary Gas Exchange; Swine

In an effort to understand the interaction between acute postural fluid shifts and hypoxia on hormonal regulation of fluid homeostasis, the authors measured the responses to head-down tilt with and without acute exposure to normobaric hypoxia. Plasma atrial natriuretic peptide (ANP), cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), plasma aldosterone (ALD), and plasma renin activity (PRA) were measured in six healthy male volunteers who were exposed to a head-down tilt protocol during normoxia and hypoxia. The tilt protocol consisted of a 17 degrees head-up phase (30 minutes), a 28 degrees head-down phase (1 hour), and a 17 degrees head-up recovery period (2 hours, with the last hour normoxic in both experiments). Altitude equivalent to 14,828 ft was simulated by having the subjects breathe an inspired gas mixture with 13.9% oxygen. The results indicate that the postural fluid redistribution associated with a 60-minute head-down tilt induces the release of ANP and cGMP during both hypoxia and normoxia. Hypoxia increased cGMP, cAMP, ALD, and PRA throughout the protocol and significantly potentiated the increase in cGMP during head-down tilt. Hypoxia had no overall effect on the release of ANP, but appeared to attenuate the increase with head-down tilt. This study describes the acute effects of hypoxia on the endocrine response during fluid redistribution and suggests that the magnitude, but not the direction, of these changes with posture is affected by hypoxia.

Topics: Adult; Aldosterone; Atrial Natriuretic Factor; Body Fluids; Cyclic AMP; Cyclic GMP; Head; Homeostasis; Hormones; Humans; Hypoxia; Male; Posture; Renin

Hypoxic pulmonary hypertension complicates many primary respiratory and cardiac conditions. To define the potential role of endothelial nitric oxide (NO) further in both the acute and chronic forms of this disorder, we determined the effects of acute changes in O2 in vitro and prolonged variations in O2 in vivo on endothelial NO production in rat main pulmonary arteries. NO production was assessed by measuring segment cyclic GMP synthesis, which was dependent on the presence of the endothelium and on NO synthase and soluble guanylate cyclase activity. With an acute decrease in pO2 in vitro from 150 to 40 mm Hg, basal endothelial NO production was attenuated by 52%. NO production stimulated by acetylcholine (ACh) or A23187, however, was not altered, suggesting that the underlying mechanism involves acute changes in endothelial intracellular calcium homeostasis or in the production or action of a local activator of endothelial NO synthase. Although prolonged hypoxia in vivo (7 days) also caused a 52% decrease in basal endothelial NO production, ACh- and A23187-stimulated production were diminished as well, by 69 and 73%, respectively; the attenuation in NO production was evident when tested at high pO2 in vitro, was not altered by exogenous L-arginine, and was reversed by 3 days of normoxic recovery, indicating that the chronic process may involve diminished availability of cofactor(s) required for NO synthase activity. Parallel studies of aortic segments showed that these effects are specific to the pulmonary endothelium. Thus, both acute and prolonged hypoxia selectively attenuate pulmonary endothelial NO production by different mechanisms.

Topics: Acetylcholine; Amino Acid Oxidoreductases; Animals; Arginine; Calcimycin; Cyclic GMP; Dose-Response Relationship, Drug; Endothelium, Vascular; Hypoxia; In Vitro Techniques; Male; Nitric Oxide; Nitric Oxide Synthase; Oxygen Consumption; Pulmonary Artery; Rats; Rats, Sprague-Dawley

The vascular response to hypoxia in endotoxin (lipopolysaccharide; LPS)-exposed rat pulmonary artery (PA) and thoracic aorta (AO) was investigated and the mechanism of the observed hypoxic responses defined. In isometric tension studies, LPS-treated AO and PA rings, with and without endothelium, demonstrated decreased (P < 0.05) contractile response to phenylephrine (PE EC50), and the dose response was shifted to the right (P < 0.01) compared with non-LPS treated rings. Both vessel types responded to hypoxia with a markedly increased (P < 0.01) and sustained (P < 0.01) constriction when preexposed to LPS. Control non-LPS rings with endothelium intact had a transient vasoconstriction in early hypoxia, which was abolished with removal of the endothelium. N omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase, increased the PE EC50 tension in LPS-treated rings, markedly reduced the duration and magnitude of the hypoxic vasoconstriction in LPS-treated rings, and attenuated the transient vasoconstriction seen in endothelium-intact, non-LPS rings (all P < 0.05). L-Arginine reversed the L-NAME effects. Hypoxia decreased guanosine 3',5'-cyclic monophosphate (cGMP) content 54 +/- 4% in all LPS and 33 +/- 4% in the non-LPS intact rings (P < 0.05). L-NAME reduced cGMP content 90 +/- 5% in all LPS rings. Indomethacin inhibited formation of a constriction factor in aortic LPS-treated rings (P < 0.01) that was endothelium dependent and unaffected by the presence of L-NAME.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Oxidoreductases; Animals; Aorta; Arginine; Cyclic GMP; Cycloheximide; Dose-Response Relationship, Drug; Endotoxins; Enzyme Induction; Hypoxia; In Vitro Techniques; Indomethacin; Nitric Oxide; Nitric Oxide Synthase; Phenylephrine; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Vasoconstriction

Neutral endopeptidase (NEP) inhibition is thought to blunt hypoxic pulmonary hypertension by reducing atrial natriuretic peptide (ANP) metabolism, but this hypothesis has not been confirmed. We measured NEP activity, guanosine 3',5'-cyclic monophosphate (cGMP) production, plasma ANP levels, and cardiac ANP synthesis in rats given an orally active NEP inhibitor (SCH-34826) during 3 wk of hypoxia. Under normoxic conditions, SCH-34826 had no effect on plasma ANP levels but reduced pulmonary and renal NEP activity by 50% and increased urinary cGMP levels (60 +/- 6 vs. 22 +/- 4 pg/mg creatinine; P < 0.05). Under hypoxic conditions, SCH-34826-treated rats had lower plasma ANP levels (1,259 +/- 361 vs. 2,101 +/- 278 pg/ml; P < 0.05), lower right ventricular systolic pressure (53 +/- 5 vs. 73 +/- 2 mmHg; P < 0.05), lower right ventricle weight-to-left ventricle+septum weight ratio (0.47 +/- 0.04 vs. 0.53 +/- 0.03; P < 0.05), and less muscularization and percent medial wall thickness of peripheral pulmonary arteries (22 +/- 5 vs. 45 +/- 8% and 17 +/- 1 vs. 25 +/- 1%, respectively; P < 0.05 for all values) than did rats treated with vehicle alone. These values were not affected by SCH-34826 under normoxic conditions. SCH-34826 decreased right ventricular ANP tissue levels in hypoxic rats (27 +/- 10 vs. 8 +/- 1 ng/mg protein; P < 0.05) but did not affect steady-state ANP mRNA levels. We conclude that NEP inhibition blunts pulmonary hypertension without increasing plasma ANP levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Chronic Disease; Cyclic GMP; Dioxolanes; Dipeptides; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Imidazoles; Male; Muscle, Smooth, Vascular; Myocardium; Neprilysin; Pyrazines; Rats; Rats, Sprague-Dawley; RNA, Messenger

Concentration of blood proteins, alpha-amino nitrogen, cGMP and endothelin-1,2 was studied in rat's blood at nitric hypoxia. The injection of NaNO2 (5 mg/100 g body weight) was followed by decrease in total protein, albumin and hemoglobin content and by sharp increase in methemoglobin and Hb-NO complexes concentration in rat's blood. Simultaneously, the elevation of free amino acids and peptides with m.w. 2500 D in blood was discovered. One of these peptides was the endothelin-1,2, whose concentration increased twice in plasma at nitric hypoxia. The increase in endothelin and cGMP concentrations is mostly linked with activation of compensative mechanisms, arising as a response to high concentrations of nitro compounds and NO in rats.

Topics: Adaptation, Physiological; Animals; Blood Proteins; Chromatography, High Pressure Liquid; Cyclic GMP; Electron Spin Resonance Spectroscopy; Endothelins; Hypoxia; Male; Rats; Rats, Wistar; Sodium Nitrite

We determined whether inhaling low levels of nitric oxide (NO) gas could selectively reverse hypoxic pulmonary vasoconstriction in the near-term newborn lamb and whether vasodilation would be attenuated by respiratory acidosis. To examine the mechanism of air and NO-induced pulmonary vasodilation soon after birth, we measured plasma and lung cGMP levels in the newly ventilated fetal lamb. Breathing at FIO2 0.10 nearly doubled the pulmonary vascular resistance index in newborn lambs and decreased pulmonary blood flow primarily by reducing left-to-right blood flow through the ductus arteriosus. Inhaling 20 ppm NO at FIO2 0.10 completely reversed hypoxic pulmonary vasoconstriction within minutes. Maximum pulmonary vasodilation occurred during inhalation of > or = 80 ppm NO. Breathing 8% CO2 at FIO2 0.10 elevated the pulmonary vascular resistance index to a level similar to breathing at FIO2 0.10 without added CO2. Respiratory acidosis did not attenuate pulmonary vasodilation by inhaled NO. In none of our studies did inhaling NO produce systemic hypotension or elevate methemoglobin levels. Four minutes after initiating ventilation with air in the fetal lamb lung, cGMP concentration nearly doubled without changing preductal plasma cGMP concentration. Ventilation with 80 ppm NO at FIO2 0.21 increased both lung and preductal plasma cGMP concentration threefold. Our data suggest that inhaled NO gas is a rapid and potent selective vasodilator of the newborn pulmonary circulation with an elevated vascular tone due to hypoxia and respiratory acidosis that acts by increasing lung cGMP concentration.

Topics: Acidosis, Respiratory; Administration, Inhalation; Animals; Animals, Newborn; Cyclic GMP; Hemodynamics; Hypoxia; Models, Biological; Nitric Oxide; Pulmonary Artery; Pulmonary Circulation; Sheep; Vascular Resistance; Vasoconstriction

We investigated the roles of the endothelium in the hypoxic responses of the isolated main pulmonary artery (PA) in the rat. Hypoxia was induced by gassing an organ chamber with 95% N2 + 5% CO2 (PO2 = 34.6 +/- 3.1 Torr) instead of 16% O2 + 5% CO2 + balance N2 (PO2 = 92.8 +/- 3.0 Torr). Vascular rings were precontracted with 2 x 10(-8) M phenylephrine. A transient hypoxic contraction and a subsequent relaxation were observed in the endothelium-intact rings. The hypoxic contraction was reduced in the endothelium-denuded rings. In contrast, there were no significant differences between the hypoxic relaxation in the endothelium-intact and endothelium-denuded rings. Inhibitors of endothelium-derived relaxing factor (EDRF) activity, 2 x 10(-6) M NG-monomethyl-L-arginine (L-NMMA) and 10(-6) M methylene blue, produced 53% and 66% reductions in hypoxic contraction, respectively, Furthermore, the amount of cyclic GMP in the endothelium-intact PA rings which had been precontracted with phenylephrine decreased from 2.10 +/- 0.45 pmol/mg protein during normoxia to 0.90 +/- 0.18 pmol/mg protein during hypoxia. Indomethacin and OKY-046 did not influence hypoxic contraction or relaxation. These results suggest that hypoxic contraction of the isolated pulmonary artery in the rat is partially induced by inhibition of the release of EDRF.

Topics: Animals; Arginine; Cyclic GMP; Endothelium, Vascular; Hypoxia; Male; Nitric Oxide; omega-N-Methylarginine; Pulmonary Artery; Rats; Rats, Sprague-Dawley

1. Previous studies in our laboratory revealed the presence of atrial natriuretic peptide (ANP) in preneural chemosensory type I cells of the cat carotid body, and demonstrated that submicromolar concentrations of the peptide inhibited carotid sinus nerve (CSN) activity evoked by hypoxia. In the present study, we have evaluated the role of the cyclic nucleotide second messenger, cyclic GMP (cGMP), and the involvement of type I cells in rabbit chemosensory inhibition. 2. Submicromolar concentrations of the potent ANP analogue, APIII, greatly elevated both the content and release of cGMP from the carotid body. Denervation experiments confirmed earlier immunocytochemical studies which suggested that APIII-induced cGMP production occurs almost exclusively in type I cells; these experiments also indicate that both the sympathetic and sensory innervation to the carotid body exert a trophic influence on the metabolism of this second messenger. 3. Submicromolar concentrations of APIII inhibited the CSN activity evoked by hypoxia (79.8 +/- 3.2% (mean +/- S.E.M.) inhibition with 100 nM APIII) and nicotine (74.5 +/- 3.6% inhibition with 100 nM APIII), but did not affect basal CSN activity established in 100% O2-equilibrated superfusion solutions. 4. The biologically inactive analogue of ANP, C-ANP, failed to produce CSN inhibition; however, the inhibitory effects of APIII were mimicked by cell-permeant analogues of cGMP (dibutyryl-cGMP and 8-bromo-cGMP, 2 mM), which likewise did not alter basal CSN activity. Because we found that unmodified cGMP was an ineffective inhibitor of CSN activity, our data suggest that APIII inhibition is mediated intracellularly by cGMP produced within the type I cells. 5. APIII does not inhibit the CSN activity produced by 20 mM K+ (in zero Ca2+ media), which very probably results from direct depolarization of the sensory nerve terminals. 6. Catecholamine release from the carotid body evoked by hypoxia is likewise not altered by APIII (100 nM). 7. The data are consistent with the notion that APIII and analogues of cGMP alter the release of excitatory and/or inhibitory transmitters from chemosensory type I cells in the carotid body.

Topics: Animals; Atrial Natriuretic Factor; Carotid Body; Chemoreceptor Cells; Cyclic GMP; Denervation; Depression, Chemical; Dibutyryl Cyclic GMP; Dose-Response Relationship, Drug; Hypoxia; Nicotine; Peptide Fragments; Potassium; Rabbits

Our laboratory has previously described in isolated 1- to 4-mm calf pulmonary arteries, an endothelium-independent contraction to hypoxia that appears to involve the removal of a H2O2-elicited guanosine 3',5'-cyclic monophosphate (cGMP)-mediated relaxation. In this study, we examined the effects of changes in O2 tension (PO2) on isolated endothelium-intact and endothelium-denuded calf pulmonary resistance arteries of approximately 200 microns in diameter. Resistance arteries precontracted with U46619 were found to undergo a contraction when exposed to a PO2 of 24-27 Torr (hypoxia) from a Po2 of 150 Torr (O2 atmosphere). This contraction was significantly larger in endothelium-intact than endothelium-removed arteries. In the intact artery, 30 microM nitro-L-arginine (NLA), an inhibitor of the biosynthesis of nitric oxide-like activators of guanylate cyclase, increased tone under O2 atmosphere and reduced the contraction to hypoxia to the level observed in the endothelium-removed artery. Reoxygenation caused a relaxation, which was not dependent on the endothelium or inhibited by NLA. The inhibitor of guanylate cyclase activation, LY83583 (10 microM), increased tone under O2 atmosphere, eliminated the contraction to hypoxia, and inhibited the relaxation to reoxygenation, whereas indomethacin (10 microM) did not alter these responses. Thus modulation of a cGMP mechanism, not involving the endothelium or metabolism of arginine, is a primary mediator of responses to changes in O2 tension, and the endothelium appears to cause an enhancement of the contraction to hypoxia via suppression by hypoxia of the tonic generation of an arginine-derived relaxing factor.

Topics: Acetylcholine; Aminoquinolines; Animals; Arginine; Cattle; Cyclic GMP; Endothelium, Vascular; Hypoxia; Indomethacin; Nitroarginine; Oxygen; Partial Pressure; Prostaglandin Antagonists; Pulmonary Artery; Vascular Resistance; Vasoconstriction; Vasodilation

We investigated cellular mechanisms that may be involved in controlling cytosol calcium and pulmonary artery pressure during hypoxia and normoxia in isolated blood-perfused ferret lungs. Alveolar hypoxia in ferret lungs causes an active increase in pulmonary vascular resistance. Hypoxic pulmonary vasoconstriction directly correlates with extracellular calcium ([Ca2+]o), and the absence of [Ca2+]o in the perfusate markedly attenuates the hypoxemia-induced pulmonary vasoconstriction. Alveolar hypoxia does not potentiate the production of thromboxane B2 (TxB2) or 6-keto-PGF1 alpha. Vanadate, a widely used inhibitor of Ca2+ATPases, increases pulmonary arterial pressure (Ppa) in the presence or absence of [Ca2+]o and without affecting the production of TxB2 or 6-keto-PGF1 alpha. Vanadate and ouabain, an inhibitor of Na+/K+ATPase, produce synergistic increases in Ppa. Amiloride, an inhibitor of Na+/Ca2+ exchange, reverses the increase in Ppa caused by ouabain, but not the increase caused by vanadate. The additional effect produced by ouabain on Ppa after near maximal vanadate effect and the ability of amiloride to reverse the pulmonary vasoconstriction caused by ouabain, but not vanadate, suggests that vanadate does not inhibit Na+/K+ATPase in ferret lungs. In addition, cyclic GMP (cGMP), which has been reported to increase the activity of Ca2+ATPases in vascular smooth muscle, was able to reverse and prevent the effect of vanadate on Ppa, but not the effect of ouabain. Inhibition of Ca2+ATPases with vanadate in ferret lungs increases pulmonary vascular resistance during both normoxia and hypoxia. The Ca2+ entry mediated by alveolar hypoxia appears to overpower the ability of Ca2+ATPases and other membrane Ca2+ transport proteins to translocate [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Calcium; Calcium-Transporting ATPases; Cyclic AMP; Cyclic GMP; Ferrets; Hypoxia; Lung; Male; Pulmonary Artery; Thromboxane B2; Vanadates; Vasoconstriction

The initiating event in hypoxic pulmonary hypertension is felt to be sustained hypoxic vasoconstriction, ultimately leading to vascular remodeling and fixed pulmonary hypertension. During the initial vasospastic phase endogenous vasodilatory pathways may serve to ameliorate the development of pulmonary hypertension. However, various studies in the systemic and pulmonary circulations have shown that chronic hemodynamic stress alters both endothelial and smooth muscle cell function. The effect of chronic hypoxia in rats was therefore tested on three major vasodilatory pathways: 1) endothelium-dependent relaxation (using endothelium-derived relaxing factor agonists and antagonists); 2) smooth muscle cell cyclic nucleotide-mediated relaxation [using guanosine and adenosine 3',5'-cyclic monophosphate (cGMP and cAMP) agonists]; and 3) ion channel-dependent relaxation (using K+ channel agonists and Ca2+ channel antagonists). It was found that short-term exposure (72 h) to hypoxia caused augmentation of K+ and Ca2+ channel-dependent relaxation with no effect on endothelium-dependent or cyclic nucleotide-mediated relaxation. More prolonged exposure (4-5 wk) was additionally associated with inhibition of endothelium-dependent relaxation and smooth muscle cell cGMP-mediated relaxation. There was no effect on either basal modulation of tone by the endothelium, cAMP-mediated relaxation, or systemic vessel relaxation. It is concluded that an early response to hemodynamic stress in the pulmonary circulation is alteration in smooth muscle cell ion channel function and/or Ca2+ homeostasis.

Topics: Animals; Calcium; Calcium Channels; Chronic Disease; Cyclic AMP; Cyclic GMP; Endothelium, Vascular; Extracellular Space; Hypoxia; Male; Potassium Channels; Pulmonary Artery; Rats; Rats, Inbred Strains; Vasoconstriction; Vasodilator Agents

We have demonstrated previously that in response to hypoxia, isolated rat pulmonary arteries show an initial endothelium-dependent relaxation followed by an endothelium-independent transient contraction. In the presence of increased extracellular Ca2+, both of these responses were enhanced in endothelium-intact arteries. Nitro-L-arginine, a blocker of the biosynthesis of endothelium-derived relaxing factor (EDRF), abolished the initial endothelium-dependent relaxation and Ca(2+)-induced enhancement of hypoxic contraction in endothelium-intact arteries but did not alter responses in endothelium-denuded vessels. Inhibition of prostaglandin formation with indomethacin had no effect on the hypoxia-elicited responses. Preincubation with LY 83583, an inhibitor of guanylate cyclase activation, abolished the initial hypoxia-elicited relaxation and subsequent contraction. M & B 22948, a guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase inhibitor, decreased tone under O2 but not under N2, causing an apparent enhancement of the contraction to hypoxia. Thus the modulation of hypoxic responses by the endothelium is dependent on changes in EDRF production, and a decrease in smooth muscle cGMP not involving an EDRF mechanism appears to mediate the endothelium-independent hypoxic contraction observed in the isolated rat pulmonary artery.

Topics: Aminoquinolines; Animals; Arginine; Cyclic GMP; Hypoxia; In Vitro Techniques; Indomethacin; Male; Nitroarginine; Pulmonary Artery; Purinones; Rats; Rats, Inbred Strains

Topics: Animals; Coronary Vessels; Cyclic GMP; Hypoxia; In Vitro Techniques; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroglycerin; Swine

Topics: Animals; Arginine; Cyclic GMP; Endothelium, Vascular; Hypoxia; In Vitro Techniques; Muscle Contraction; Muscle, Smooth, Vascular; omega-N-Methylarginine; Pulmonary Artery; Rats

Hypoxia is a potent coronary-vasodilating signal; its mechanisms are still controversial. We have assessed the possible role of nitric oxide (NO) in hypoxic coronary vasodilatation (HCVD) in isolated guinea pig hearts perfused at constant pressure. HCVD was elicited by a 1-minute 100% N2 exposure; coronary flow doubled within 1 minute of hypoxia (early phase) and returned to baseline within 40 seconds after reoxygenation (late phase). The early phase of HCVD was associated with a rapid approximately eightfold increase in cGMP overflow, an indication of NO release. The specific NO synthase inhibitor N omega-methyl-L-arginine (NMA, 0.1-1 mM) antagonized HCVD and the associated increase in cGMP spillover (maximum inhibition, approximately 65%); excess arginine (1.2 mM) prevented both effects. The late phase of HCVD was associated with an increase in adenosine overflow and was attenuated by the adenosine receptor antagonist BW A1433 (1 microM; maximum inhibition, approximately 45%). Indomethacin (10 microM) inhibited HCVD in spontaneously beating hearts by approximately 35% but had no effect in hearts paced at faster rates. NMA and BW A1433 were more effective in combination than alone (maximum inhibition, approximately 72%). However, irrespective of the concentrations used, there was no synergism among the anti-HCVD effects of NMA, BW A1433, and indomethacin, nor was HCVD completely inhibited by the antagonists, whether alone or in combination. Our findings indicate that NO is an important mediator of the early phase of HCVD, whereas additional mechanisms and/or factors, including adenosine and vasodilatatory prostaglandins, contribute to the late phase.

Topics: Adenosine; Animals; Arginine; Coronary Vessels; Cyclic GMP; Guinea Pigs; Hypoxia; In Vitro Techniques; Indomethacin; Male; Nitric Oxide; Vasodilation; Xanthines

We have examined the effects of exposing rats to hypoxia (10% fractional inspired O2 concentration) for 2 and 7 days on endothelium-dependent and -independent vasodilation and also on the ability of guanosine 3',5'-cyclic monophosphate (cGMP) to activate cGMP-dependent protein kinase (G-kinase) in rat conduit pulmonary arteries (PA). The ability of acetylcholine (ACh) and sodium nitroprusside (SNP) to both relax PA rings and elevate tissue cGMP levels was significantly attenuated in PA from hypoxic animals. The ability of atrial natriuretic peptide to relax and generate cGMP in PA rings was unchanged by hypoxia. Relaxation and elevation of cGMP levels induced by SNP in aortic rings was unaltered by hypoxia. Similarly, hypoxia did not alter the concentration-dependent activation by exogenous cGMP of G-kinase. We conclude that chronic exposure of rats to hypoxia results in a selective impairment of soluble guanylyl cyclase in rat PA, leading to an attenuation of ACh- and SNP-induced cGMP accumulation and relaxation.

Topics: Animals; Aorta; Chronic Disease; Cyclic GMP; Endothelium, Vascular; Guanylate Cyclase; Hypoxia; Male; Nitroprusside; Phenylephrine; Pulmonary Artery; Rats; Rats, Wistar; Solubility; Vasodilation; Vasodilator Agents

This experiment was designed to investigate whether chronic hypoxia affect rat pulmonary artery (PA) endothelium-dependent relaxation and the content of cGMP in PA. Both ACh and ATP could induce endothelium-dependent relaxation of PA, not prevented by indomethacin, but completely abolished by methylene blue. These results indicated that vasodilatation of PA induced by both ACh and ATP is mediated by EDRF (endothelium-derived relaxing factor). Chronic hypoxia significantly depressed PA endothelium-dependent relaxation. The percent relaxation of IPPA and EPPA by 10(-6) mol/L ACh was 61.3% and 59.2% of those in control, and the percent relaxation of IPPA and EPPA by 1.8 x 10(-5) mol/L ATP was 64.9% and 55.3% respectively of the control. Chronic hypoxia also depressed SNP-induced endothelium-independent relaxation. Chronic hypoxia significantly decreased the content of cGMP in PA. The basic level of cGMP was 51.9 +/- 5.7 (n = 14) in hypoxia group and 84.9 +/- 9.7 (n = 14) pmol/g wet wt. in control group (P less than 0.01). After treatment of PA with ACh (10(-7) mol/L), the content of cGMP was 91.4 +/- 7.3 (n = 5) pmol/g wet wt. in hypoxic group and 240.8 +/- 30.6 (n = 5) pmol/g wet wt. in control group (P less than 0.01). Our data suggest that chronic hypoxia might depress rat pulmonary artery endothelium-dependent relaxation through the inhibition of soluble guanylate cyclase in vascular smooth muscle cells.

Topics: Animals; Cyclic GMP; Endothelium, Vascular; Hypoxia; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Pulmonary Artery; Rats; Rats, Inbred Strains

Guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) are mediators of smooth muscle relaxation. In this study, selective inhibitors of phosphodiesterase (PDE) isozymes were used to assess the role of cyclic nucleotide hydrolysis in angiotensin II (ANG II) and hypoxic pulmonary vasoconstriction. In isolated rat lungs, the hypoxic pressor response (HPR) was induced with a 95% N2-5% CO2 gas mixture. When administered during the plateau of the HPR, trequinsin (nonselective PDE inhibitor) and indolidan (cGMP-inhibitable cAMP PDE inhibitor) significantly (P = 0.01) decreased the pulmonary arterial pressure (Ppa) by 60 +/- 7 and 53 +/- 3%, respectively, compared with zaprinast (cGMP PDE inhibitor), rolipram (cGMP-insensitive cAMP PDE inhibitor), and the 0.1% dimethyl sulfoxide (DMSO) vehicle control, which decreased the Ppa by 6 +/- 3, 4 +/- 3, and 0%, respectively. In the trequinsin and indolidan groups, the subsequent ANG II pressor responses and HPRs were significantly (P = 0.01) decreased when compared with the zaprinast, rolipram, and DMSO groups. During normoxia, none of the PDE inhibitor (0.3-30 microM) had an effect on the baseline Ppa. These results suggest that cAMP hydrolysis by the cGMP-inhibitable cAMP PDE play a significant role in pulmonary vascular tone regulation.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Angiotensin II; Animals; Blood; Blood Pressure; Cyclic GMP; Hypoxia; In Vitro Techniques; Male; Perfusion; Phosphodiesterase Inhibitors; Pulmonary Circulation; Rats; Vasoconstriction

The effect of severe hypoxia in quiescent or contracted (prostaglandin F2 alpha) canine coronary artery rings with and without endothelium was studied. Hypoxia induced an initial transient relaxation followed by a sustained contraction. The hypoxic contraction in quiescent rings was comparable in rings with and without endothelium. The facilitation of the contraction to prostaglandin F2 alpha was more pronounced in rings with endothelium. Increasing the level of contractions by augmenting the contraction of prostaglandin F2 alpha potentiated the hypoxic contraction in rings with endothelium only. Methylene blue, LY 83583, and nitro-L-arginine reversed the hypoxic facilitation in contracted rings into relaxation, whereas M&B 22948 augmented it. In quiescent coronary preparations, methylene blue reversed the hypoxic contraction into relaxation in preparations with and without endothelium, whereas nitro-L-arginine had the same effect in vessels with endothelium only. SIN-1, nitroglycerin, and dibutyryl guanosine 3',5'-cyclic monophosphate (cGMP) unmasked hypoxic facilitation in rings without endothelium. This was not observed with isoproterenol. The measurement of the level of cGMP revealed an increased level in rings with endothelium compared with those without endothelium under control oxygenation. This difference disappeared during hypoxia due to a decrease of cGMP content in vessels with endothelium. The results suggest that a moderate increase of the cGMP level in vascular smooth muscle is a prerequisite for the occurrence of hypoxia-induced facilitation in contracted canine coronary arteries.

Topics: Animals; Arginine; Coronary Vessels; Cyclic GMP; Dinoprost; Dogs; Endothelium, Vascular; Female; Hypoxia; In Vitro Techniques; Male; Methylene Blue; Muscle Contraction; Muscle, Smooth, Vascular; Nitric Oxide; Nitroarginine; Oxygen

To test the hypothesis that exogenous atrial natriuretic peptide (ANP) prevents the acute pulmonary pressor response to hypoxia, ANP (20-micrograms/kg bolus followed by 1-microgram.kg-1.min-1 infusion) or vehicle was administered intravenously to conscious rats beginning 3 min before exposure to hypoxia or room air for 90 min. Exogenous ANP abolished the acute pulmonary pressor response to hypoxia in association with marked and parallel increases in plasma ANP and guanosine 5'-cyclic monophosphate (cGMP) and with a significant increase in lung cGMP content. To examine whether endogenous ANP modulates the acute pulmonary pressor response to hypoxia, rats were pretreated with a monoclonal antibody (Ab) to ANP and exposed to hypoxia. Mean pulmonary arterial pressure (MPAP) in the Ab-treated rats was not different from control over the first 6 h of hypoxic exposure. Thereafter, the Ab-treated group had significantly higher MPAP than control. Our data suggest that 1) exogenous ANP blocks the pulmonary pressor response to acute hypoxia via stimulation of cGMP accumulation in the pulmonary vasculature, and 2) endogenous ANP may modulate the subacute, but not acute, phase of hypoxic pulmonary hypertension.

Topics: Acute Disease; Animals; Antibodies, Monoclonal; Atrial Natriuretic Factor; Blood Pressure; Cyclic GMP; Heart Rate; Hypertension, Pulmonary; Hypoxia; Lung; Male; Pulmonary Artery; Radioimmunoassay; Rats; Rats, Inbred Strains

This experiment was designed to determine whether chronic hypoxia affects endothelium-dependent relaxation and cGMP content of rat pulmonary artery (PA). Both Ach and ATP were found to induce endothelium-dependent relaxation of PA; and this relaxation was not prevented by indomethacin, but was completely abolished by methylene blue. Chronic hypoxia significantly depressed the endothelium-dependent relaxation: the relaxation responses of intra-PA (IPA) and extra-PA (EPA) to 10(-6) mol/L Ach in the hypoxic group were 61.3% and 59.2% of those in control, and the relaxation responses of IPA and EPA to 1.8 x 10(-5) mol/L ATP in the hypoxic group were 64.9% and 55.2% of those in the control, respectively. Chronic hypoxia significantly decreased the basic level and Ach-induced accumulation of cGMP in the PA. Our data suggest that chronic hypoxia might depress rat pulmonary artery endothelium-dependent relaxation through the inhibition of cytosolic soluble guanylate cyclase in vascular smooth muscle cells.

Topics: Acetylcholine; Adenosine Triphosphate; Animals; Cyclic GMP; Endothelium, Vascular; Hypoxia; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Pulmonary Artery; Rats; Rats, Inbred Strains

ANP increases cellular cGMP content in cultured hepatocytes and decreases Ca2(+)-inflow in a concentration- and time-dependent manner which explains a beneficial effect on hypoxia cell injury (25). Both observations are mimicked by SNP and 8-Br-cGMP and blocked by Ly 83583 indicating a cGMP-mediated mechanism. The protective effect was also inhibited by Pertussis Toxin (PT) without lowering the elevated cGMP-level. But PT in combination with ANP leads to a higher Ca2(+)-inflow. Stimulated Na(+)-inflows are also be lowered by ANP. Here, neither SNP can mimick nor PT can inhibit this effect. Our results now indicate that the beneficial effect by ANP at the cellular level is mediated through cGMP which decreases calcium-inflow. ANP seems to control Ca2(+)-channels direct via a PT-sensitive G-protein and indirect by a cGMP-mediated mechanism and Na(+)-channels cGMP-independent through a PT-insensitive G-protein, thus preventing cells on hypoxia and oxygen radicals.

Topics: Aminoquinolines; Animals; Atrial Natriuretic Factor; Calcium; Cells, Cultured; Cyclic GMP; Free Radicals; GTP-Binding Proteins; Hypochlorous Acid; Hypoxia; Kinetics; Liver; Nitroprusside; Pertussis Toxin; Rats; Virulence Factors, Bordetella

We have recently suggested that relaxation of isolated precontracted intrapulmonary arteries from calves to H2O2 or O2 may involve the activation of guanylate cyclase by peroxide metabolism via catalase. In this study, ethanol, an agent that modulates peroxide metabolism by catalase and selectively inhibits the activation of guanylate cyclase by H2O2 but not by nitric oxide-related activators, was employed to further investigate the role of catalase in pulmonary arterial relaxation and guanylate cyclase activation by O2 and H2O2. In precontracted pulmonary arteries, ethanol reverses H2O2-elicited relaxation and increases in guanosine 3',5'-cyclic monophosphate (cGMP) tissue levels without affecting similar responses to nitroprusside. The pulmonary arteries employed in this study show a hypoxic contraction that is associated with decreases in cGMP levels, and reoxygenation produces a somewhat phasic relaxation and a marked increase in cGMP levels. Ethanol produces an O2 tension-dependent contraction and reverses relaxation to reoxygenation associated with inhibition of O2-elicited increases in cGMP levels. Thus ethanol appears to function as a mimic of hypoxia by inhibiting relaxations elicited by O2. These findings support a hypothesized role for H2O2-dependent activation of guanylate cyclase in O2-dependent regulation of pulmonary arterial smooth muscle tone.

Topics: Animals; Cattle; Cyclic GMP; Ethanol; Guanylate Cyclase; Hydrogen Peroxide; Hypoxia; In Vitro Techniques; Oxygen; Pulmonary Artery; Vasoconstriction; 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

We have previously reported that the isolated rat branch pulmonary artery (PA) contracts when made hypoxic and that the contraction is dependent in large part on the presence of a functioning endothelium. This study tested if the hypoxic contraction was caused by reduced endothelium-derived relaxing factor (EDRF) activity. To do so we tested if chemical inhibitors of EDRF mimicked the effect of hypoxia, if PA guanosine 3',5'-cyclic monophosphate (cGMP) fell during hypoxic contraction, and if stimulation of smooth muscle cGMP attenuated hypoxic contraction. We found that the EDRF inhibitors hemoglobin and methylene blue caused a concentration-dependent increase in PA force that equaled that produced by hypoxia. PA cGMP decreased in endothelium-intact rings from 105 +/- 14 pM/g (wet wt) during normoxia to 41 +/- 9 pM/g during hypoxia. In endothelium-denuded rings normoxic cGMP was reduced to 32 +/- 10 pM/g with no further decrease during hypoxia. The endothelium-independent stimulators of cGMP, nitric oxide, and 8-bromo-cGMP, reduced maximum hypoxic contraction by 80 +/- 11 and 93 +/- 3%, respectively, whereas the endothelium-dependent stimulator acetylcholine did not. PA adenosine 3',5'-cyclic monophosphate (cAMP) fell only slightly during hypoxia and cAMP inhibitors failed to mimic the hypoxic contraction. We conclude that the hypoxic contraction of isolated rat PA is caused largely by decreased EDRF activity.

Topics: Acetylcholine; Adenosine Monophosphate; Animals; Cyclic AMP; Cyclic GMP; Dideoxyadenosine; Endothelium, Vascular; Hemoglobins; Hypoxia; In Vitro Techniques; Isomerism; Kinetics; Male; Methylene Blue; Muscle, Smooth, Vascular; Nitric Oxide; Phenylephrine; Pulmonary Artery; Rats; Rats, Inbred Strains; Thionucleotides; Vasodilation

The aim of the study was to investigate whether atrial natriuretic peptides have a physiological role in regulation of the pulmonary circulation.. Plasma concentrations of immunoreactive atrial natriuretic peptide and guanosine-3',5'-cyclic monophosphate (cGMP) were measured during evaluation of pulmonary vascular tone by multipoint pulmonary arterial pressure-cardiac index (Ppa/Q) relationships.. Experimental animals were 17 mongrel dogs of either sex, 21-35 kg weight, anaesthetised with pentobarbitone.. Measurements of Ppa/Q relationships and atrial natriuretic peptide/cGMP were made during hyperoxia (Fio2 0.4) and hypoxia (Fio2 0.1). Hypoxic pulmonary vasoconstriction, defined as hypoxia induced increase in pulmonary artery pressure over the entire range of Q studied from 2-5 litre.min-1.m-2, was elicited in nine dogs ("responders"). In the other eight dogs, hypoxia did not change pulmonary artery pressure over the entire range of Q studied ("non-responders"). At neither the highest nor the lowest Q in hyperoxia did atrial natriuretic peptide and cGMP concentrations differ between these two groups, nor did acute reduction in Q affect the concentrations in either group. At the highest Q, plasma atrial natriuretic peptide increased in hypoxia from 11(SEM 2) to 15(3) pmol.litre-1 in the responders (p less than 0.05), and from 15(2) to 20(2) pmol.litre-1 in the non-responders (p less than 0.05). However at the lowest Q, atrial natriuretic peptide was increased in non-responders only, from 17(3) to 23(4) pmol.litre-1 (p less than 0.05). CGMP did not vary significantly in any experimental condition.. Hypoxia slightly increased plasma atrial natriuretic peptides without any relationship with associated pulmonary haemodynamic changes. These data do not support the hypothesis that atrial natriuretic peptides play a physiological role in the regulation of the pulmonary circulation in dogs.

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Cardiac Output; Cyclic GMP; Dogs; Female; Hypoxia; Male; Pulmonary Circulation; Vasoconstriction

The present studies were conducted to examine the role of cerebrovascular guanylate cyclase in hypoxic cerebral vasodilatation. In arteries mounted in vitro for measurements of isometric tension, 20 min of hypoxia (bath oxygen partial pressure, approximately 15 Torr) significantly increased cyclic GMP levels from 16 to 32, from 15 to 25 and from 20 to 38 pmol/g in rabbit common carotid, internal carotid and basilar arteries. These increases were blocked either by pretreatment with 3 microM methylene blue, or by removal of the vascular endothelium. Methylene blue also significantly delayed hypoxic relaxation in the basilar and internal carotid arteries, and blocked transient hypoxic vasoconstriction in the common carotid. Together, these in vitro results demonstrate that vascular cytosolic guanylate cyclase participates in an endothelium-dependent manner in the direct effects of hypoxia on cerebral arteries, and that the nature of this participation varies significantly between arteries. When methylene blue (20 mg/kg) was administered in vivo, however, it had no effect on the magnitude of hypoxic cerebral vasodilatation as determined by both local (mass spectrometry) and global (venous outflow) methods of blood flow measurement. This latter finding suggests that: 1) large and small cerebral arteries may differ significantly in terms of either endothelial function or sensitivity to methylene blue; or 2) feedback regulation of other mechanisms of hypoxic cerebral vasodilatation compensate for the effects of guanylate cyclase inhibition. Additional experiments using other inhibitors of cytosolic guanylate cyclase and/or vessels isolated from the cerebral microcirculation will be necessary to distinguish between these possibilities.

Topics: Animals; Cerebrovascular Circulation; Cyclic GMP; Drug Interactions; Hypoxia; Injections, Intravenous; Male; Methylene Blue; Muscle, Smooth, Vascular; Nitroglycerin; Papaverine; Rabbits; Vasodilation

We have reported evidence that endothelium-independent relaxations of isolated bovine pulmonary arteries to H2O2 and to reoxygenation with 95% O2-5% CO2 after brief exposure to N2 (5% CO2) appear to be mediated by the activation of guanylate cyclase via H2O2 metabolism through catalase. Treatment of endothelium-removed pulmonary arteries with a potential guanylate cyclase-inhibitor, LY 83583, or with the inhibitor of the Zn+2, Cu+2-superoxide dismutase (SOD) diethyldithiocarbamic acid (DETCA), antagonized guanosine 3',5'-cyclic monophosphate (cGMP)-associated relaxation to H2O2, to reoxygenation and to glyceryl trinitrate, but not the adenosine 3',5'-cyclic monophosphate-associated relaxation to isoproterenol. Superoxide anion (O2-.) levels, detected by lucigenin-elicited chemiluminescence, were enhanced by LY 83583 or DETCA treatment of pulmonary arteries at ambient PO2. Chemiluminescence produced by LY 83583 was markedly potentiated by DETCA treatment, decreased at addition of exogenous SOD, and inhibited markedly by anoxia. LY 83583, but not DETCA, stimulated cyanide-insensitive O2 consumption, consistent with redox cycling of the compound independent of mitochondrial respiration. We propose that O2-. generated on the metabolism of LY 83583, or from cellular electron donors after SOD inhibition by DETCA, inhibits cGMP-mediated relaxations of pulmonary arteries.

Topics: Aminoquinolines; Animals; Anions; Cattle; Cyclic GMP; Ditiocarb; Hydrogen Peroxide; Hypoxia; Isoproterenol; Nitroglycerin; Oxygen; Oxygen Consumption; Pulmonary Artery; Superoxides; Vasodilation

We examined, in isolated blood perfused rat lungs, the effect of the cell permeable 8-bromo derivative of cGMP on pulmonary vasoconstriction induced by either alveolar hypoxia or angiotensin II. 8-Bromo cGMP dose-dependently reduced both hypoxia-(IC50 = 2.2 X 10(-5) M) and angiotensin-II-induced pulmonary vasoconstriction (IC50 = 5.0 X 10(-5) M). This effect of 8-bromo cGMP on pulmonary vasoconstriction was not affected by cyclooxygenase blockade. M & B 22948 (0.1 mM), an inhibitor of cGMP-phosphodiesterase, reduced synergistically with 8-bromo cGMP the hypoxia or angiotensin-II-induced vasoconstriction. The cGMP-phosphodiesterase inhibitor M & B 22948, by itself, selectively reduced hypoxia-induced vasoconstriction, suggesting a modulating effect of endogenous cGMP during hypoxic vasoconstriction.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Angiotensin II; Animals; Cyclic GMP; Dose-Response Relationship, Drug; Hypoxia; Lung; Male; Meclofenamic Acid; Purinones; Rats; Vasoconstriction

Hyperbaric oxygenation (HBO) and pyridinolcarbamat were mainly applied in the treatment of elderly patients with peptic ulcer attended by coronary heart disease and atherosclerosis. To evaluate the condition of intracellular regulators after the treatment, a study was made of the time-course of changes in the content of cyclic nucleotides in blood plasma and gastric mucosa. The use of HBO in the treatment of peptic ulcer raised the efficacy of the multimodality therapy and accelerated the epithelization of erosive and ulcerative defects. The use of pyridinolcarbamat turned to produce a beneficial therapeutic effect and to be protective with respect to the gastric mucosa. Thus it was found desirable to apply the drug to the treatment of ulcers mainly occurring in the stomach. The changes in the content of cyclic nucleotides in blood plasma and gastric mucosa may play an important role in the realization of positive metabolic changes in gastric mucosa, induced by pyridinolcarbamat and HBO in peptic ulcer patients.

Topics: Aged; Anti-Ulcer Agents; Carbamates; Combined Modality Therapy; Cyclic AMP; Cyclic GMP; Female; Gastric Mucosa; Humans; Hyperbaric Oxygenation; Hypoxia; Male; Middle Aged; Peptic Ulcer; Pyridinolcarbamate

The effect of hypoxia on endothelium-dependent and endothelium-independent vasodilation was studied in phenylephrine-precontracted, isolated rings of rabbit first-branch pulmonary artery. Concentration-dependent relaxation responses to the endothelium-dependent dilators methacholine, ATP, and the calcium ionophore (A23187) as well as to the endothelium-independent dilators sodium nitroprusside and isoproterenol were obtained before, during, and after exposure to hypoxia (PO2 = 42 +/- 1 mm Hg) in the presence of indomethacin (2.8 x 10(-5) M). This moderate degree of hypoxia inhibited (p less than 0.05) endothelium-dependent but not endothelium-independent relaxation responses without producing irreversible vascular damage. In parallel experiments, cyclic GMP accumulation in pulmonary vascular rings in response to maximal doses of the above vasodilators was measured in the presence and absence of hypoxia. Cyclic GMP accumulation in response to endothelium-dependent dilators (methacholine, ATP, and A23187) was inhibited (p less than 0.05) by hypoxia while cyclic GMP accumulation in response to the endothelium-independent dilator sodium nitroprusside was not. When phenylephrine precontracted vessels were exposed to hypoxia in the absence of vasodilators, a small, transient increase in tension occurred, which was greater in endothelium-intact than in endothelium-denuded vessels (0.70 +/- 0.12 vs. 0.09 +/- 0.03 g, respectively; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Animals; Calcimycin; Cyclic GMP; Endothelium, Vascular; Hypoxia; Isoproterenol; Methacholine Chloride; Methacholine Compounds; Nitric Oxide; Nitroprusside; Pulmonary Artery; Rabbits; Receptors, Purinergic; Vasodilation

The present experiments measured cAMP and cGMP in the arterial chemosensory tissue of the rabbit carotid body exposed for 10 min in vitro to normoxic or hypoxic conditions, or to specific activators of adenylate cyclase (forskolin) and guanylate cyclase (sodium nitroprusside). The enzyme activators elevated the basal levels of cAMP (48 x) and cGMP (3.7 x), respectively. Hypoxic media increased cAMP in the carotid body by 3.6-fold, but the levels of cGMP were reduced by 33% in media equilibrated with low O2. The data are consistent with the notion that cyclic nucleotides are involved in the transduction of natural stimuli and/or the neurotransmitter feedback modulation of chemosensory type I cells.

Topics: Action Potentials; Animals; Carotid Body; Colforsin; Cyclic AMP; Cyclic GMP; Hypoxia; In Vitro Techniques; Nitroprusside; Rabbits; Second Messenger Systems; Synaptic Transmission

Topics: Animals; Biological Products; Cyclic GMP; Hypoxia; In Vitro Techniques; Methylene Blue; Nitric Oxide; Pulmonary Artery; Rats; Vasoconstriction

The effect of endothelium on hypo-oxygenation--induced reactions of preactivated smooth muscle (SM) of the canine and porcine large coronary arteries, was studied. Decreased oxygenation of Krebs--bicarbonate solution (PO2-26.6-19.9 gPa) resulted in biphasic changes in the preparation with intact endothelium: a considerable relaxation was preceded by transient contractions. After removal of the endothelium a transient increase of tension was reduced or absent. Blockade of prostaglandins, leukotrienes and free radicals did not affect hypo-oxygenation-induced contraction of the VSM. The data suggest that endothelium can modulate the response of the coronary arteries SM to decrease in oxygen tension. Endothelium-dependent transient contraction during hypo-oxygenation is mediated through the increase of cGMP production.

Topics: Animals; Coronary Vessels; Cyclic GMP; Dogs; Endothelium, Vascular; Hypoxia; In Vitro Techniques; Muscle Contraction; Muscle, Smooth, Vascular; Oxygen Consumption; Partial Pressure; Swine

Topics: Animals; Biological Transport, Active; Calcium; Carotid Arteries; Cerebral Arteries; Cyclic GMP; Hypoxia; Kinetics; Rabbits

An in vivo brain perfusion technique was used to examine effects of hypoxia on cerebral cortical metabolism in barbiturate-anesthetized rats. Dulbecco's phosphate-buffered solution (PBS), or Dulbecco's PBS + 6 mM glucose, was infused into the right carotid circulation for 0 to 3 min, at a rate that reduced regional cerebral blood flow to the ipsilateral parietal lobe by more than 40% and O2 delivery by about 50%. The duration of infusion of either solution was correlated negatively with the ipsilateral parietal lobe concentrations of glucose, ATP, and phosphocreatine (PCr), and positively with parietal concentrations of lactate and cAMP. cGMP increased in relation to infusion duration of Dulbecco's PBS. Statistically significant elevations of brain lactate occurred after 1 min of infusion of Dulbecco's PBS; lactate was elevated and glucose was reduced after 2 min of infusion of either solution. Brain ATP, PCr, and glycogen concentrations decreased in relation to the elevation in brain lactate, and the [PCr]:[ATP] ratio declined. The results demonstrated that limited hypoxia stimulated cerebral glycolysis and produced a concurrent decrease in brain ATP and PCr. However, ATP was spared to a degree, at the expense of PCr.

Topics: Adenosine Triphosphate; Animals; Brain; Carotid Arteries; Cerebrovascular Circulation; Cyclic AMP; Cyclic GMP; Glucose; Glycogen; Hypoxia; Lactates; Male; Oxygen; Parietal Lobe; Perfusion; Phosphocreatine; Rats; Rats, Inbred Strains

We investigated a hypothesis that an oxygen sensor involved in hypoxia-induced relaxation of vascular smooth muscle may reside in endothelial cells. We also determined the oxygen dependence of hypoxia-induced decreases in cyclic guanosine 3',5'-monophosphate concentrations in vascular smooth muscle rings. Rings of canine femoral artery, rabbit thoracic aorta, and lamb ductus arteriosus, either with an intact endothelium or with damaged or absent endothelium, were studied using organ baths that allowed changes in PO2 without a change in pH. Hypoxia-induced relaxations of rabbit thoracic aorta, lamb ductus arteriosus, and canine femoral artery were not dependent on an intact endothelium. The magnitude of hypoxia-induced relaxations was unchanged in rings of canine femoral artery without intact endothelium compared to rings with endothelium. Quasi-steady state organ bath PO2-mechanical tension relationships were unchanged in rings of canine femoral artery without endothelium over an organ PO2 range of 200-20 mm Hg. With rabbit thoracic aorta, magnitudes of hypoxia-induced relaxations were significantly smaller in rings without endothelium. Quasi-steady state plots, where mechanical tension was given as percentage of maximal relaxation, were similar in rings either with or without intact endothelium. Cyclic guanosine 3',5'-monophosphate concentrations were shown to be oxygen-sensitive, decreasing during hypoxia-induced relaxations with a threshold PO2 of 80-100 mm Hg with canine femoral artery, and 60-80 mm Hg with rabbit thoracic aorta rings, but this finding seems unrelated to the mechanism of hypoxia-induced relaxation.

Topics: Animals; Carbachol; Cyclic GMP; Dogs; Ductus Arteriosus; Endothelium; Female; Hypoxia; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Oxygen; Partial Pressure; Rabbits; Sheep

10(-6) M cAMP were shown to induce a 61% and 21% increase in 45Ca binding to sarcolemma proteins in intact and injured (circulatory hypoxia) hearts, respectively. The addition of exogenous protein kinase equalized 45Ca-binding levels in normal and impaired sarcolemma. The decrease in 45Ca-binding capacity by 16 and 36% was detected in the presence of 10(-7) and 10(-6) M of cGMP, respectively. In impaired hearts, cAMP and Ca-ATPase activity levels remain constant, while cGMP content increases, as compared to normal myocardial level.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Cyclic AMP; Cyclic GMP; Dogs; Hypoxia; Myocardium; Protein Binding; Protein Kinases; Sarcolemma

A study was made of the effects of isothiobarbamine and guthimine (10 and 50 mg/kg, respectively) on the content of cAMP and cGMP in the brain cortex (BC) and hippocamp under normal conditions and hypoxia. Isothiobarbamine did not change the content of both cyclic nucleotides under normoxia, whereas under hypoxia it reduced the level of the cyclic nucleotides in the BC and raised it in the hippocamp. Guthimine increased their content in the BC and did not change it in the hippocamp under normoxia, whereas under hypoxia it increased the cAMP content in the hippocamp and did not change it in the BC. The cGMP content descended in both the structures under study.

Topics: Animals; Brain; Cerebral Cortex; Cyclic AMP; Cyclic GMP; Drug Evaluation, Preclinical; Guanylthiourea; Hippocampus; Hypoxia; Male; Rats; Rats, Inbred Strains; Thiopental; Thiourea

Topics: Animals; Cyclic AMP; Cyclic GMP; Histamine; Hypertension, Pulmonary; Hypoxia; Swine

The effects of 8-bromo-cAMP (10(-4)M) and 8-bromo-cGMP (10(-4)M) on tissue lactate, NADH, creatine phosphate (CP), ATP, ADP and AMP were studied in hypoxic (50% oxygen saturation) spontaneously beating rat atria. CP/ATP ratio and energy charge (EC) were also calculated. In hypoxic rat atria there was a significant increase in tissue lactate, NADH, ADP and AMP and a decrease in CP, CP/ATP ratio and EC. 8-bromo-cGMP abolished these hypoxia-induced changes. The levels of lactate and NADH decreased and those of CP, ATP, ADP and EC increased after administration of 8-bromo-cGMP. 8-bromo-cAMP increased the level of lactate during hypoxia, but did not affect the other hypoxia-induced changes. The present work indicates that cyclic GMP can change the redox state and energy state in a more favourable direction for the hypoxic rat heart.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenine Nucleotides; Animals; Cyclic GMP; Energy Metabolism; Heart Atria; Hypoxia; Lactates; Lactic Acid; Male; Myocardium; NAD; Oxidation-Reduction; Phosphocreatine; Rats; Rats, Inbred Strains

The effect of anoxia or 2,4-dinitrophenol (DNP) on the phosphorylase activity and the cyclic AMP and the cyclic GMP content was studied in smooth muscle preparations. When the aerobic conditions were changed to anaerobic in experiments on bovine mesenteric artery, there was a significant increase in the activity of phosphorylase a during the first 60 min. We had observed a reduction of the glycogen content of the artery during this time period, which accounted for about 2/3 of the increase in lactate production (Pasteur effect). Under anaerobic conditions the content of cyclic AMP in the vessel was not changed, and the increase in phosphorylase a activity was not inhibited by a blockade of adrenergic beta-receptors. DNP, which like anoxia inhibits the mitochondrial production of ATP, increased the phosphorylase a activity to the same extent as anoxia. Anoxia and DNP also enhanced the activity of phosphorylase a in pig thoracic aorta and rabbit colon smooth muscle. In thoracic aorta both anoxia and DNP produced a more transient and smaller increase in the phosphorylase a activity than in the mesenteric artery. The Pasteur effect was also relatively smaller (100%) in thoracic aorta than in mesenteric artery (400%). It is suggested that an anoxic increase in the phosphorylase a activity participates in the Pasteur effect in smooth muscle.

Topics: 2,4-Dinitrophenol; Adrenergic beta-Antagonists; Animals; Aorta, Thoracic; Cattle; Colon; Cyclic AMP; Cyclic GMP; Dinitrophenols; Hypoxia; In Vitro Techniques; Mesenteric Arteries; Muscle, Smooth, Vascular; Phosphorylase a; Phosphorylases; Swine

Sodium nitroprusside (NP) was found to decrease the tissue lactate level under conditions of high (HiOxSa) and low (hypoxia) oxygen saturation in spontaneously beating rat atria. Nitroprusside also increased the level of cyclic GMP (cGMP) in a dose-dependent manner in both states. The time course and dose dependence of these effects suggest that cGMP might be responsible for the decreased tissue level of lactate demonstrated. The findings suggest that cGMP might inhibit lactate production in HiOxSa and antagonize the hypoxia-induced acceleration of anaerobic metabolism. It is proposed that a state of low-rate glycolysis results from this inhibitory effect of cGMP. Furthermore, the possible role of cGMP as a feed-back regulator of the redox state is discussed in the light of these findings.

Topics: Animals; Cyclic GMP; Dose-Response Relationship, Drug; Ferricyanides; Glycolysis; Hypothermia; Hypoxia; In Vitro Techniques; Lactates; Lactic Acid; Male; Myocardial Contraction; Myocardium; Nitroprusside; Rats; Rats, Inbred Strains

Topics: Acetylcholine; Animals; Bucladesine; Cats; Cyclic GMP; Electric Conductivity; Electrophysiology; Heart; Hypoxia; In Vitro Techniques; Isoproterenol; Myocardial Contraction; Nucleotides, Cyclic

In order to study the influence of hypoxia on cyclic nucleotides in the brain, we reduced arterial Po(2) for 15-30 min in lightly anaesthetised and artificially ventilated rats to obtain values ranging from about 45 to about 10 mm Hg. In an additional group (arterial Po(2) 18-22 mm Hg), the tissue hypoxia was aggravated by moderate arterial hypotension (mean arterial blood pressure about 80 mm Hg). In all animals, electrocortical activity was recorded. Cyclic GMP concentrations in cerebral cortex were unchanged in all groups but one. In that group, in which tissue hypoxia was severe enough to induce a suppression-burst EEG pattern and a measurable reduction in the adenylate energy charge, cyclic GMP concentrations were slightly increased (p less than 0.05). Cyclic AMP concentrations remained unaltered at all degrees of hypoxia studied. It is concluded that changes in cyclic nucleotides in brain tissue occur first at such severe degrees of hypoxia of the duration studied that function and metabolism are profoundly altered.

Topics: Animals; Brain; Cyclic AMP; Cyclic GMP; Electroencephalography; Hypotension; Hypoxia; Male; Oxygen; Partial Pressure; Rats

Topics: Acetylcholine; Animals; Atropine; Blood Pressure; Calcium; Coronary Disease; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Hypoxia; Male; Myocardial Contraction; Perfusion; Purine Nucleotides; Rats; Rats, Inbred Strains

A possible involvement of cyclic nucleotides (cAMP and cGMP) in the regulation of cardiac contractility and glycolysis during hypoxia was examined in spontaneously beating rat atria. A reduction of the high oxygen saturation (HiOxSa) of the incubation medium from 95-100% to half produced a rapid decline of the amplitude. The deterioration of 50% was seen after 30 sec. of hypoxia. The decline was partly antagonized by noradrenaline (NA, 1 X 10(-6) M) or hypercalcaemia (5.7 X 10(-3) M instead of 1.9 X 10(-3) M). The cAMP level remained unchanged during the first 12 min. of hypoxia, but the cGMP content increased gradually and reached a significantly increased level in 4-8 min. Paradoxically, the production of lactate decreased, after 30 sec. of hypoxia, but accelerated then 2-4 min. after the onset of hypoxia. The depletion of creatine phosphate and ATP stores was initiated after 2 min. of hypoxia. The arterial content of the active forms of phosphofructokinase and lactate dehydrogenase gradually rose during hypoxia. Sodium nitroprusside (SNP, 1 X 10(-4) M) and NA produced increases in cGMP and cAMP levels, respectively, both in HiOxSa and hypoxia. SNP induced a slight and NA a marked increase in the amplitude in HiOxSa. Verapamil (1 X 10(-6) M) decreased the contractility, but did not affect the levels of cAMP and cGMP. Both SNP and verapamil decreased the lactate production, but they could not resist the NA-induced increase in the atrial lactate level. Hypercalcaemia increased the amplitude but slightly reduced the lactate production in HiOxSa. 45Ca-uptake was reduced to about 35 per cent of control as measured between 5 and 10 min. of hypoxia. It is concluded that the lack of oxygen could have direct and parallel effects on the sarcolemma and on the mitochondria. The former could result in the deterioration of contractility and the latter in the termination of aerobic energy production. Cyclic nucleotides are not involved in either of these phenomena. However, at the low rate of anaerobic glycolysis, e.g. in HiOxSa or at the very early stage of hypoxia, cGMP could inhibit and cAMP accelerate the lactate production.

Topics: Animals; Cyclic AMP; Cyclic GMP; Glycolysis; Hypoxia; Lactates; Lactic Acid; Male; Myocardial Contraction; Nitroprusside; Norepinephrine; Rats; Rats, Inbred Strains; Verapamil

Hepatic glycogen metabolism in aerobic and hypoxic conditions has been assessed with respect to glycogenolysis, phosphorylase alpha activity and nucleotide content. Insulin did not inhibit glycogen breakdown nor stimulate lipogenesis in the aerobic perfused liver. Partial ischaemia induced glycogen breakdown, release of glucose and changes in nucleotide content in the perfused liver. Phosphorylase alpha content increased within 2 min in response to total ischaemia, in vivo and in the perfused liver. This change was paralleled by an increase in hepatic AMP. Glycogen synthase alpha activity decreased, as did the hepatic content of both cyclic AMP and cyclic GMP.

Topics: Adenine Nucleotides; Aerobiosis; Animals; Carbohydrate Metabolism; Cyclic AMP; Cyclic GMP; Glycogen Synthase; Hypoxia; In Vitro Techniques; Ischemia; Liver Glycogen; Male; Phosphorylase a; Rats

Topics: Animals; Calcium; Cats; Coronary Disease; Cyclic AMP; Cyclic GMP; Haplorhini; Heart Diseases; Hypoxia; Myocardial Contraction; Myocardium; Papio; Rats; Swine

In vitro and in vivo studies showed that methylation of homologous DNA in nuclear homogenates of rat bone marrow is controlled by cyclic nucleotides and some hormones. The cyclic nucleotides and their dibutyryl analogs inhibited homologous methylation of DNA in the presence and absence of phosphodiesterase inhibitors. In the presence of Ca2+ the inhibiting effect of the nucleotides was more pronounced. Prostaglandins inhibited DNA methylation in a weaker degree while insulin and erythropoietin had a stronger effect in comparison with the nucleotides. Histamine stimulated DNA methylation, whereas acute hypoxic hypoxia caused a reduction in the rate of DNA methylation.

Topics: Animals; Bone Marrow; Bucladesine; Calcium; Cell Nucleus; Cyclic AMP; Cyclic GMP; Dibutyryl Cyclic GMP; DNA; DNA (Cytosine-5-)-Methyltransferases; Erythropoietin; Hypoxia; Insulin; Kinetics; Methylation; Methyltransferases; Rats

The ability of endogenous myocardial catecholamines to participate in the development of myocardial cellular alterations during a short period of severe hypoxia (30 min) was studied in isolated, Langendorff-perfused rat heart preparation, arrested by a high potassium concentration (16 mM) and perfused in the absence of exogenous substrate (Table I). Tyramine, which accelerated catecholamine depletion, also increased myocardial cell damage as assessed by a higher lactate dehydrogenase (LDH) release and a more marked reduction in cellular levels of high energy phosphates and glycogen (Table II). On the other hand, under conditions of beta-blockade (atenolol), hypoxia-induced tissular damage was reduced (Table II). These changes could be related to modifications in the cellular content of cyclic AMP (cAMP) since cAMP was consistently higher during the first 30 min of hypoxic perfusion than in control normoxic hearts (Table III) whereas cyclic GMP content remained unchanged. Moreover, interventions increasing cellular content of cAMP (theophylline, dibutyryl-cAMP) also increased hypoxic damage (Table IV), whereas N-methyl imidazole which reduced cellular content of cAMP lessened hypoxia-induced cellular alterations (Table IV). It is concluded that cellular lesions developing during the first 30 min of hypoxia in isolated arrested rat heart preparation perfused without exogenous substrate could be related to intracellular accumulation of cAMP occurring under the effect of endogenous catecholamine release.

Topics: Adenosine Triphosphate; Animals; Atenolol; Catecholamines; Cyclic AMP; Cyclic GMP; Glycogen; Hypoxia; L-Lactate Dehydrogenase; Male; Myocardium; Perfusion; Phosphocreatine; Rats; Time Factors; Tyramine

Topics: Animals; Cyclic AMP; Cyclic GMP; DNA; Female; Hypoxia; Lung; Rats; Thymidine; Time Factors; Tritium

Topics: Animals; Cyclic AMP; Cyclic GMP; Hypoxia; Lung; Pulmonary Circulation; Swine; Vascular Resistance; Vasoconstriction

Topics: Acetylcholine; Animals; Brain; Cyclic AMP; Cyclic GMP; Glucose; Hypoxia; Male; Mice; Potassium Cyanide; Sodium Nitrite

The isolated isovolumic rat heart was used as a model of cardiac hypoxia. Force of cardiac contraction and cardiac cyclic nucleotide levels (cyclic GMP and cyclic AMP) were monitored in hearts subjected to hypoxia for 5 min and allowed to recover by reoxygenation. Hearts were obtained from both control animals and animals pretreated with methylprednisolone at 18 hr and 1 hr prior to sacrifice. Myocardial levels of cyclic GMP which were significantly (p less than 0.05) elevated above control during all periods of hypoxia were found to be lower when hearts were pretreated with methylprednisolone prior to hypoxic exposure. Hearts of animals pretreated with methylprednisolone also demonstrated better recovery during reoxygenation than did control hearts. These studies suggest that methylprednisolone may be beneficial in the prevention of myocardial failure following hypoxia via a modulation in myocardial cyclid GMP content.

Topics: Animals; Coronary Disease; Cyclic AMP; Cyclic GMP; Disease Models, Animal; Hypoxia; In Vitro Techniques; Male; Methylprednisolone; Myocardial Contraction; Myocardium; Rats

Topics: Animals; Atmospheric Pressure; Chronic Disease; Cyclic AMP; Cyclic GMP; Female; Histamine; Hypoxia; Lung; Methylprednisolone; Rats

The effect of three different levels of 24-h hypobaric hypoxia (630 mm Hg, 520 mm Hg, and 340 mm Hg) on rat lung cAMP and cGMP was studied. Liver was also examined to evaluate comparatively with lung. Lung cAMP concentration averaged 33.6 +/- 2.2 (SE) pmol/mg protein, and cGMP averaged 2.4 +/- 0.21 (SE) pmol/mg protein. Twenty-four-hour hypoxia resulted in a significant 40% decrease in lung cAMP at 520 and 340 mm Hg. The magnitude of change was the same for both hypoxic levels suggesting threshold dependency. In contrast, liver cAMP was not affected by the hypoxic exposure. Lung and liver cGMP were also relatively refractory to hypoxia. The decreased cAMP concentration seen in the hypoxic lung returned to normal level within 24 hours. These data show that acute hypoxia has a selective action on lung cAMP.

Topics: Animals; Cyclic AMP; Cyclic GMP; Hypoxia; Liver; Lung; Male; Rats; Time Factors

Gastric fundic mucosae in vitro from four species of frog and Necturus secrete HCO-3 at a steady-state rate of 0.25-0.55 microneq-cm-2-h-1 which corresponds to 5-10% of maximal H+ secretion. Net alkalinization was quantitated in mucosae with spontaneously resting H+ secretion or in mucosae inhibited by histamine H2-receptor antagonists or SNC-. HCO-3 secretion was inhibited by DNP (10(-4) M), CN- (10(-2) M), or anoxia. Acetazolamide inhibited alkalinization at 10(-2) M when added to the nutrient side and at 10(-4) M on the luminal side. Carbachol (10(-4) M) and DBcGMP (10(-4) M) stimulated alkalinization and caused a transient rise in the transmucosal PD; DBcAMP (10(-3) M) was without effect. An almost identical secretion occurred spontaneously in antral mucosae and was insensitive to histamine (10(-5) M). Occurrence in both antral and fundic mucosa suggests that active alkalinization is a property of gastric surface epithelial cells. Gastric alkalinization may protect the luminal surface of the mucosa from the damaging effects of acid and contribute to the continuous removal of H+ ions from gastric contents.

Topics: Acetazolamide; Animals; Anura; Bicarbonates; Bucladesine; Carbachol; Cyanides; Cyclic GMP; Diffusion; Dinitrophenols; Gastric Mucosa; Histamine; Histamine H2 Antagonists; Hydrogen; Hydrogen-Ion Concentration; Hypoxia; Membrane Potentials; Ranidae; Thiocyanates; Urodela

Topics: Acid-Base Equilibrium; Adenosine Triphosphate; Animals; Blood; Brain; Carbon Dioxide; Cerebellum; Cerebral Cortex; Cyclic AMP; Cyclic GMP; Halothane; Hydrogen-Ion Concentration; Hypoxia; Lactates; Mice; Oxygen; Phosphocreatine; Pyruvates; Spinal Cord

Topics: Adaptation, Physiological; Animals; Atmosphere Exposure Chambers; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Hypoxia; Male; Mice; Neurotransmitter Agents; Periodicity; Rats; Receptors, Adrenergic; Receptors, Cholinergic

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenosine Monophosphate; Animals; Choroid Plexus; Cyclic AMP; Cyclic GMP; Hypoxia; In Vitro Techniques; Kinetics; Male; Nitrogen; Ouabain; Pia Mater; Rabbits; Temperature; Theophylline

Isolated rat hearts were subjected to hypoxic perfusion on a recirculating Langendorff apparatus. Following a 30-min-period of aerobic stabilization the hearts were perfused for 30 min with media equilibrated with 84% N2, 12%O2 and 4% CO2. At the end of the hypoxic period myocardial concentrations of cyclic AMP and cyclic GMP were determined by radioimmunoassay. Exposure to hypoxia resulted in a significant increase in cyclic AMP (p less than 0.01) and a decrease in cyclic GMP (p less than 0.05) as compared to hearts perfused for 60 min with media gassed with 96% O2. 4% CO2.

Topics: Animals; Cyclic AMP; Cyclic GMP; Hypoxia; Male; Myocardium; Perfusion; Rats

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Calcium; Carotid Body; Cyclic AMP; Cyclic GMP; Enzyme Activation; Hypoxia; Kinetics; Male; Phosphoric Diester Hydrolases; Rats

Isolated rat hearts were perfused with hormonal concentrations of glucagon during a hypoxic perfusion to determine whether it would enhance recovery after reoxygenation. Rat hearts were divided into two groups: (1) those perfused with glucose-free Tyrode's solution and (2) those perfused with Tyrode's solution containing glucose. During 3 minutes of exposure to hypoxia both untreated hearts and hearts perfused with glucagon demonstrated a decrease in contractile force to 10-20% of control. When glucose was present in the perfusion medium, cardiac performance was better during both the periods of hypoxia and reoxygenation. During reoxygenation, recovery of contractile force was significantly better (P less than 0.05) in glucagon-perfused hearts than in untreated hearts; this improved recovery occurred regardless of whether glucose was included in the medium. The enhanced recovery of the glucagon-perfused hearts was associated with decreases in myocardial levels of guanosine, 3',5'-monophosphate (cyclic GMP) both during the periods of hypoxia and reoxygenation. At the end of the hypoxic period, cyclic GMP levels in the glucagon-perfused hearts were 20-64% of the levels in untreated hearts. Similarly, after 5 minutes of reoxygenation cyclic GMP levels in the glucagon-perfused hearts were 21% of the levels in the untreated hearts. The effect of glucagon on adenosine 3',5'-monophosphate (cyclic AMP) concentrations in untreated hearts and in hearts receiving glucagon was not significantly different either after 3 minutes of hypoxia or during reoxygenation. The rate of anaerobic glycolysis after 3 minutes of hypoxia was higher in untreated hearts than in glucagon-perfused hearts, as determined by the lactate content of coronary perfusates. These studies suggest that hormonal concentrations of glucagon exert a protective effect on the hypoxic rat heart which involves a modulation of cardiac cyclic GMP accumulation.

Topics: Animals; Cyclic AMP; Cyclic GMP; Glucagon; Glycolysis; Hypoxia; Male; Myocardial Contraction; Myocardium; Oxygen Consumption; Rats

Topics: Animals; Bone Marrow; Bone Marrow Cells; Cells, Cultured; Cyclic AMP; Cyclic GMP; Erythropoietin; Female; Humans; Hypoxia; Mice; Mice, Inbred Strains; Rabbits; Spleen; Time Factors

Topics: Animals; Cyclic AMP; Cyclic GMP; Fasting; Hypoxia; Liver; Lung; Male; Rats

Topics: Animals; Cyclic AMP; Cyclic GMP; Erythropoiesis; Hypoxia; Iron Radioisotopes; Mice; Time Factors

Topics: Adrenal Glands; Animals; Carbon Dioxide; Corticosterone; Cyclic AMP; Cyclic GMP; Denervation; Hypoxia; Male; Organ Size; Pentobarbital; Phentolamine; Propranolol; Rats; Spleen; Theophylline; Tritium