15-hydroxy-11-alpha-9-alpha-(epoxymethano)prosta-5-13-dienoic-acid and Hypoxia

Preeclampsia (PE) is a pregnancy-related disorder characterized by hypertension and proteinuria that affects 3-10% of all pregnancies. Although its pathophysiology remains obscure, placental hypoxia-induced oxidative stress and alterations in vascular function, morphology, and endothelial barrier integrity are considered to play a key role in the development of preeclampsia. In this study, placental villous explants of noncomplicated placentae and BeWo cells were subjected to hypoxia. The effect of placental hypoxic-conditioned medium (HCM) on intraluminal-induced contraction and endothelial barrier integrity in chorionic arteries was investigated using pressure myography. The impact of BeWo cell HCM on endothelial cell viability, reactive oxygen species formation and inflammation was also determined. Alterations in arterial morphology and contractile responsiveness to the thromboxane A2 analog (U46619) after exposure to placental HCM were examined immunohistochemically and by wire myography, respectively. Intraluminal administration of placental HCM induced vasoconstriction and increased the endothelial permeability for KCl, which was concentration-dependently prevented by quercetin. Placental and BeWo cell HCMs decreased endothelial cell viability, increased the production of reactive oxygen species and enhanced the secretion of IL-6 and IL-8. The cross-sectional area of the arterial media was increased upon exposure to placental HCM, which was associated with increased vascular proliferation and contractile responsiveness to U46619, and all of these effects were prevented by the antioxidants quercetin and RRR-α-tocopherol. This study is the first to comprehensively demonstrate the link between factors secreted by placental cells in response to hypoxia and vascular abnormalities and paves the way for new diagnostic approaches and therapies to better protect the maternal vasculature during and after a preeclampsia-complicated pregnancy.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Cell Line; Culture Media, Conditioned; Endothelial Cells; Endothelium, Vascular; Female; Humans; Hypoxia; In Vitro Techniques; Permeability; Placenta; Pre-Eclampsia; Pregnancy; Reactive Oxygen Species; Vasoconstriction

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by a progressive increase in pulmonary artery pressure due to an increase in vessel tone and occlusion of vessels. The endogenous vasodilator prostacyclin and its analogs are used as therapeutic agents for PAH. However, their pharmacological effects on occlusive vascular remodeling have not been elucidated yet. Selexipag is a recently approved, orally available and selective prostacyclin receptor agonist with a non-prostanoid structure. In this study, we investigated the pharmacological effects of selexipag on the pathology of chronic severe PAH in Sprague-Dawley and Fischer rat models in which PAH was induced by a combination of injection with the vascular endothelial growth factor receptor antagonist Sugen 5416 and exposure to hypoxia (SuHx). Oral administration of selexipag for three weeks significantly improved right ventricular systolic pressure and right ventricular (RV) hypertrophy in Sprague-Dawley SuHx rats. Selexipag attenuated the proportion of lung vessels with occlusive lesions and the medial wall thickness of lung arteries, corresponding to decreased numbers of Ki-67-positive cells and a reduced expression of collagen type 1 in remodeled vessels. Administration of selexipag to Fischer rats with SuHx-induced PAH reduced RV hypertrophy and mortality caused by RV failure. These effects were probably based on the potent prostacyclin receptor agonistic effect of selexipag on pulmonary vessels. Selexipag has been approved and is used in the clinical treatment of PAH worldwide. It is thought that these beneficial effects of prostacyclin receptor agonists on multiple aspects of PAH pathology contribute to the clinical outcomes in patients with PAH.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetamides; Animals; Cell Proliferation; Collagen Type I; Disease Models, Animal; Heart Ventricles; Hemodynamics; Hypertrophy, Right Ventricular; Hypoxia; Indoles; Lung; Male; Pulmonary Arterial Hypertension; Pyrazines; Pyrroles; Rats, Sprague-Dawley; Receptors, Epoprostenol; Systole; Vascular Remodeling

Pulmonary hypertension is a severe, incurable disease with a poor prognosis. Although treatment regimens have improved during the last 2 decades, current pharmacologic strategies are limited and focus on the modulation of only a few pathways related to endothelin, NO, and prostacyclin signaling. Therefore, the identification of novel molecular targets is urgently needed. We found that the β

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adrenergic beta-2 Receptor Agonists; Animals; Blood Pressure; Hypertension, Pulmonary; Hypoxia; Lung; Mice; Phenylephrine; Pulmonary Artery; Receptors, Adrenergic, alpha-1; Terbutaline

What is the central question of this study? Hypoxic pulmonary vasoconstriction has never been characterized in isolated mouse pulmonary arteries of different generations in detail. What is the main finding and its importance? We found that only small intrapulmonary arteries (80-200 μm in diameter) exhibit hypoxic pulmonary vasoconstriction. The observed response was sustained, significantly potentiated by depolarization-induced preconstriction and not dependent on the endothelium or TRPC6 channels.. Hypoxic pulmonary vasoconstriction (HPV) is a physiological response of pulmonary arteries, which adapts lung perfusion to regional ventilation. The properties of HPV vary significantly between animal species. Despite extensive use of mouse models in studies of HPV, this physiological response has never been characterized in isolated mouse pulmonary arteries in detail. Using wire myography, we investigated the effect of 80 min exposure to hypoxia on the tone in mouse pulmonary arteries of different generations in the presence and absence of preconstriction. Hypoxia induced a sustained relaxation in non-preconstricted extrapulmonary arteries (500-700 μm in diameter), but not in the presence of KCl-induced preconstriction. Large intrapulmonary arteries (450-650 μm in diameter) did not exhibit a significant response to the hypoxic challenge. In contrast, in small intrapulmonary arteries (80-200 μm in diameter), hypoxia elicited a slowly developing sustained constriction, which was independent of the endothelium. The response was significantly potentiated in arteries preconstricted with KCl, but not with U46619. Hypoxic pulmonary vasoconstriction was not altered in pulmonary arteries of TRPC6-deficient mice, which suggests that this response corresponds to the sustained phase of biphasic HPV observed earlier in isolated, buffer-perfused and ventilated mouse lungs. In conclusion, we have established a protocol that allows the study of sustained HPV in isolated mouse pulmonary arteries. The data obtained might be useful for future studies of the mechanisms of HPV in mice.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Endothelium, Vascular; Female; Hypoxia; In Vitro Techniques; Male; Mice; Mice, Knockout; Muscle Tonus; Muscle, Smooth, Vascular; Myography; Potassium Chloride; Pulmonary Artery; Pulmonary Circulation; TRPC Cation Channels; TRPC6 Cation Channel; Vasoconstriction; Vasoconstrictor Agents

Preeclampsia is a major health problem in human pregnancy, severely complicating 5-8% of all pregnancies. The emerging molecular mechanism is that conditions like hypoxic stress trigger the release of placental messengers into the maternal circulation, which causes preeclampsia. Our objective was to develop an in vitro model, which can be used to further elucidate the molecular mechanisms of preeclampsia and which might be used to find a remedy.. Human non-complicated term placentas were collected. Placental explants were subjected to severe hypoxia and the conditioned media were added to chorionic arteries that were mounted into a myograph. Contractile responses of the conditioned media were determined, as well as effects on thromboxane-A2 (U46619) induced contractility. To identify the vasoactive compounds present in the conditioned media, specific receptor antagonists were evaluated.. Factors released by placental explants generated under severe hypoxia induced an increased vasoconstriction and vascular contractility to thromboxane-A2. It was found that agonists for the angiotensin-I and endothelin-1 receptor released by placental tissue under severe hypoxia provoke vasoconstriction. The dietary antioxidant quercetin could partially prevent the acute and sustained vascular effects in a concentration-dependent manner.. Both the acute vasoconstriction, as well as the increased contractility to U46619 are in line with the clinical vascular complications observed in preeclampsia. Data obtained with quercetin supports that our model opens avenues for e.g. nutritional interventions aimed at treating or preventing preeclampsia.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Cell Hypoxia; Chorion; Constriction, Pathologic; Culture Media, Conditioned; Endothelium, Vascular; Female; Humans; Hypoxia; Muscle Contraction; Myography; Placenta; Pre-Eclampsia; Pregnancy; Vasoconstriction; Vasoconstrictor Agents

Augmented vasoconstrictor reactivity is thought to play an important role in the development of chronic hypoxia (CH)-induced neonatal pulmonary hypertension. However, whether this response to CH results from pulmonary endothelial dysfunction and reduced nitric oxide (NO)-mediated vasodilation is not well understood. We hypothesized that neonatal CH enhances basal tone and pulmonary vasoconstrictor sensitivity by limiting NO-dependent pulmonary vasodilation. To test this hypothesis, we assessed the effects of the NO synthase (NOS) inhibitor

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Animals, Newborn; Chronic Disease; Enzyme Inhibitors; Free Radical Scavengers; Hypoxia; Lung; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Tyrosine; Vascular Resistance; Vasoconstriction; Vasoconstrictor Agents; Vasodilation

In addition to its renowned poisonous effects, carbon monoxide (CO) is being recognized for its beneficial actions on inflammatory and vasoregulatory pathways, particularly when applied at low concentrations via CO-releasing molecules (CO-RMs). In the lung, CO gas and CO-RMs are suggested to decrease pulmonary vascular tone and hypoxic pulmonary vasoconstriction (HPV). However, the direct effect of CO-RMs on the pulmonary vasoreactivity in isolated lungs has not yet been investigated. We assessed the effect of CORM-2 and CORM-3 on the pulmonary vasculature during normoxia and acute hypoxia (1% oxygen for 10 min) in isolated ventilated and perfused mouse lungs. The effects were compared with those of inhaled CO gas (10%). The interaction of CORM-2 or CO with cytochrome P-450 (CYP) was measured simultaneously by tissue spectrophotometry. Inhaled CO decreased HPV and vasoconstriction induced by the thromboxane mimetic U-46619 but did not alter KCl-induced vasoconstriction. In contrast, concentrations of CORM-2 and CORM-3 used to elicit beneficial effects on the systemic circulation did not affect pulmonary vascular tone. High concentration of CO-RMs or long-term application induced a continuous increase in normoxic pressure. Inhaled CO showed spectral alterations correlating with the inhibition of CYP. In contrast, during application of CORM-2 spectrophotometric signs of interaction with CYP could not be detected. Application of CO-RMs in therapeutic doses in isolated lungs neither decreases pulmonary vascular tone and HPV nor does it induce spectral alterations that are characteristic of CO-inhibited CYP. High doses, however, may cause pulmonary vasoconstriction.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Carbon Monoxide; Hypoxia; Lung; Mice; Mice, Inbred C57BL; Organometallic Compounds; Vasoconstriction

This study describes the effect of variable oxygen supply on relaxing responses induced by α-calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) on isolated pig coronary arteries in vitro. Organ culture during normoxia (21% of O₂) and hypoxia (5% of O₂) induced a significant leftward shift of the AM concentration-response curves compared with fresh vessels altering the pEC₅₀ values from 6.9 ± 0.04 to 8.0 ± 0.04, whereas the potency (pEC₅₀) of αCGRP was attenuated from 8.8 ± 0.04 to 7.6 ± 0.04. AM₂₂₋₅₂ exerted significant antagonistic effect on AM-induced vasorelaxation in hypoxic and normoxic conditions (apparent pK(B) = 6.8-7.2), whereas no antagonistic effect was observed in fresh and hyperoxic (95%) organ cultured vessels. The antagonistic effect exerted by αCGRP₈₋₃₇ (10⁻⁶·⁵-10⁻⁵·⁵ M) on αCGRP-induced vasodilatation in fresh vessels (derived from Schild plot pA₂ = 7.4 ± 0.1) was unaltered during organ culture. The antagonistic effect exerted by αCGRP₈₋₃₇ (10⁻⁶ M) on AM-induced vasorelaxation in fresh vessels (apparent pK(B) = 7.4 ± 0.1) was absent during hypoxic organ culture. The receptor activity-modifying proteins 1 (RAMP1)/calcitonin-like receptor (CLR) messenger RNA ratio was reduced and RAMP2/CLR messenger RNA ratio was increased during hypoxic and normoxic organ culture compared with fresh vessels. Hypoxic organ culture for 24-72 hours potentiated the AM-induced vasorelaxation through an AM₂₂₋₅₂-sensitive receptor but attenuated the vasorelaxant effect of CGRP through the CGRP receptors. This could possibly be explained by relatively decreased levels of RAMP1, thus favoring RAMP2 + CLR complex (=AM receptor) formation during hypoxic organ culture.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adrenomedullin; Animals; Calcitonin Gene-Related Peptide; Calcitonin Receptor-Like Protein; Coronary Vessels; Hypoxia; Muscle Contraction; Myocardium; Organ Culture Techniques; Peptide Fragments; Receptor Activity-Modifying Proteins; RNA, Messenger; Swine; Vasodilation; Vasodilator Agents

Hypoxia-induced pulmonary hypertension (HPH) is characterized by sustained pulmonary vasoconstriction and vascular remodeling, both of which are mediated by pulmonary artery smooth muscle cell (PASMC) contraction and proliferation, respectively. An increase in cytosolic Ca²⁺ concentration ([Ca²⁺]cyt) is a major trigger for pulmonary vasoconstriction and an important stimulus for cell proliferation in PASMCs. Ca²⁺ influx through voltage-dependent Ca²⁺ channels (VDCC) is an important pathway for the regulation of [Ca²⁺]cyt. The potential role for L- and T-type VDCC in the development of HPH is still unclear. Using a hypoxic-induced pulmonary hypertension mouse model, we undertook this study to identify if VDCC in pulmonary artery (PA) are functionally upregulated and determine which type of VDCC are altered in HPH. Mice subjected to chronic hypoxia developed pulmonary hypertension within 4 wk, and high-K⁺- and U-46619-induced contraction of PA was greater in chronic hypoxic mice than that in normoxic control mice. Additionally, we demonstrate that high-K⁺- and U-46619-induced Ca²⁺ influx in PASMC is significantly increased in the hypoxic group. The VDCC activator, Bay K8864, induced greater contraction of the PA of hypoxic mice than in that of normoxic mice in isometric force measurements. L-type and T-type VDCC blockers significantly attenuated absolute contraction of the PA in hypoxic mice. Chronic hypoxia did not increase high-K⁺- and U-46619-induced contraction of mesenteric artery (MA). Compared with MA, PA displayed higher expression of calcium channel voltage-dependent L-type α1C-subunit (Cav1.2) and T-type α1H-subunit (Cav3.2) upon exposure to chronic hypoxia. In conclusion, both L-type and T-type VDCC were functionally upregulated in PA, but not MA, in HPH mice, which could result from selectively increased expression of Cav1.2 and Cav3.2.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium; Calcium Channel Agonists; Calcium Channels, L-Type; Calcium Channels, T-Type; Chronic Disease; Gene Expression Regulation; Hypoxia; Male; Mesenteric Arteries; Mice; Potassium; Pulmonary Artery; Time Factors; Vasoconstrictor Agents; Vasodilation

Plasma hemoglobin (Hb) scavenges endothelium-derived nitric oxide (NO), producing systemic and pulmonary vasoconstriction in many species. We hypothesized that i.v. administration of murine cell-free Hb would produce pulmonary vasoconstriction and enhance hypoxic pulmonary vasoconstriction (HPV) in mice. To assess the impact of plasma Hb on basal pulmonary vascular tone in anesthetized mice we measured left lung pulmonary vascular resistance (LPVRI) before and after infusion of Hb at thoracotomy. To confirm the findings obtained at thoracotomy, measurements of right ventricular systolic pressure (RVSP) and systemic arterial pressure (SAP) were obtained in closed-chest wild-type mice. To elucidate whether pretreatment with Hb augments HPV we assessed the increase in LPVRI before and during regional lung hypoxia produced by left mainstem bronchial occlusion (LMBO) in wild-type mice pretreated with Hb. Infusion of Hb increased SAP but did not change pulmonary arterial pressure (PAP), left lung pulmonary arterial flow (QLPA) or LPVRI in either wild-type or diabetic mice with endothelial dysfunction. Scavenging of NO by plasma Hb did not alter HPV in wild-type mice. Inhibition of NO synthase with l-NAME did not change the basal LPVRI, but augmented HPV during LMBO. Our data suggest that scavenging of NO by plasma Hb does not alter pulmonary vascular tone in mice. Therefore, generation of NO in the pulmonary circulation is unlikely to be responsible for the low basal pulmonary vascular tone of mice.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Analysis of Variance; Animals; Blood Pressure; Diabetes Mellitus, Experimental; Hemoglobins; Hypoxia; Lung; Male; Mice; Mice, Inbred C57BL; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Superoxides; Thoracotomy; Vascular Resistance; Vasoconstriction; Vasoconstrictor Agents

Actin polymerization (APM), regulated by Rho GTPases, promotes myocyte force generation. Hypoxia is known to impede postnatal disassembly of the actin cytoskeleton in pulmonary arterial (PA) myocytes. We compared basal and agonist-induced APM in myocytes from PA and descending aorta (Ao), under hypoxic and normoxic conditions. We also examined effects of thromboxane challenge on force generation and cytoskeletal assembly in resistance PA and renal arteries from neonatal swine with persistent pulmonary hypertension (PPHN) induced by 72-h normobaric hypoxia, compared with age-matched controls. Synthetic and contractile phenotype myocytes from neonatal porcine PA or Ao were grown in hypoxia (10% O(2)) or normoxia (21% O(2)) for 7 days, then challenged with 10(-6) M thromboxane mimetic U46619. F/G actin ratio was quantified by laser-scanning cytometry and by cytoskeletal fractionation. Thromboxane receptor (TP) G protein coupling was measured by immunoprecipitation and probing for Gαq, G12, or G13, RhoA activation by Rhotekin-RBD affinity precipitation, and LIM kinase (LIMK) and cofilin phosphorylation by Western blot. Isometric force to serial concentrations of U46619 was measured in muscular pulmonary and renal arteries from PPHN and control swine; APM was quantified in fixed contracted vessels. Contractile PA myocytes exhibit marked Rho-dependent APM in hypoxia, with increased active RhoA and LIMK phosphorylation. Their additional APM response to U46619 challenge is independent of RhoA, reflecting decreased TP association with G12/13 in favor of Gαq. In contrast, hypoxic contractile Ao myocytes polymerize actin modestly and depolymerize to U46619. Both basal APM and the APM response to U46619 are increased in PPHN PA. APM corresponds with increased force generation to U46619 challenge in PPHN PA but not renal arteries.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Actin Cytoskeleton; Actins; Animals; Animals, Newborn; Aorta, Thoracic; Cell Culture Techniques; Cell Hypoxia; Disease Models, Animal; Humans; Hypertension, Renovascular; Hypoxia; Infant, Newborn; Laser Scanning Cytometry; Lim Kinases; Muscle Cells; Persistent Fetal Circulation Syndrome; Phosphorylation; Pulmonary Artery; Receptors, Thromboxane; Renal Artery; rhoA GTP-Binding Protein; Swine; Thromboxanes; Vasoconstriction; Vasoconstrictor Agents

Enhancement of cerebral blood flow by hypoxia is critical for brain function, but signaling systems underlying its regulation have been unclear. We report a pathway mediating hypoxia-induced cerebral vasodilation in studies monitoring vascular disposition in cerebellar slices and in intact mouse brains using two-photon intravital laser scanning microscopy. In this cascade, hypoxia elicits cerebral vasodilation via the coordinate actions of H(2)S formed by cystathionine β-synthase (CBS) and CO generated by heme oxygenase (HO)-2. Hypoxia diminishes CO generation by HO-2, an oxygen sensor. The constitutive CO physiologically inhibits CBS, and hypoxia leads to increased levels of H(2)S that mediate the vasodilation of precapillary arterioles. Mice with targeted deletion of HO-2 or CBS display impaired vascular responses to hypoxia. Thus, in intact adult brain cerebral cortex of HO-2-null mice, imaging mass spectrometry reveals an impaired ability to maintain ATP levels on hypoxia.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Triphosphate; Analysis of Variance; Animals; Blotting, Western; Carbon Monoxide; Cerebrum; Cystathionine beta-Synthase; DNA Primers; Heme Oxygenase (Decyclizing); Hydrogen Sulfide; Hypoxia; Immunohistochemistry; Mass Spectrometry; Mice; Microcirculation; Microscopy, Confocal; Regional Blood Flow; Vasodilation

Pulmonary hypertension remains a major clinical problem despite current therapies. In this study, we examine for the first time a novel pharmacological target, smooth muscle myosin, and determine if the smooth muscle myosin inhibitor, CK-2019165 (CK-165) ameliorates pulmonary hypertension.. Six domestic female pigs were surgically instrumented to measure pulmonary blood flow and systemic and pulmonary vascular dynamics. Pulmonary hypertension was induced by hypoxia, or infusion of the thromboxane analog (U-46619, 0.1 µg/kg/min, i.v.). In rats, chronic pulmonary hypertension was induced by monocrotaline.. CK-165 (4 mg/kg, i.v.) reduced pulmonary vascular resistance by 22±3 and 28±6% from baseline in hypoxia and thromboxane pig models, respectively (p<0.01 and 0.01), while mean arterial pressure also fell and heart rate rose slightly. When CK-165 was delivered via inhalation in the hypoxia model, pulmonary vascular resistance fell by 17±6% (p<0.05) while mean arterial pressure and heart rate were unchanged. In the monocrotaline model of chronic pulmonary hypertension, inhaled CK-165 resulted in a similar (18.0±3.8%) reduction in right ventricular systolic pressure as compared with sildenafil (20.3±4.5%).. Inhibition of smooth muscle myosin may be a novel therapeutic target for treatment of pulmonary hypertension.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Antihypertensive Agents; Dose-Response Relationship, Drug; Epoprostenol; Female; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Monocrotaline; Nitroprusside; Piperazines; Pulmonary Artery; Purines; Rats; Sildenafil Citrate; Smooth Muscle Myosins; Sulfones; Swine; Vascular Resistance; Vasoconstriction; Vasodilator Agents

Responses to a selective azaindole-based Rho kinase (ROCK) inhibitor (azaindole-1) were investigated in the rat. Intravenous injections of azaindole-1 (10-300 µg/kg), produced small decreases in pulmonary arterial pressure and larger decreases in systemic arterial pressure without changing cardiac output. Responses to azaindole-1 were slow in onset and long in duration. When baseline pulmonary vascular tone was increased with U46619 or L-NAME, the decreases in pulmonary arterial pressure in response to the ROCK inhibitor were increased. The ROCK inhibitor attenuated the increase in pulmonary arterial pressure in response to ventilatory hypoxia. Azaindole-1 decreased pulmonary and systemic arterial pressures in rats with monocrotaline-induced pulmonary hypertension. These results show that azaindole-1 has significant vasodilator activity in the pulmonary and systemic vascular beds and that responses are larger, slower in onset, and longer in duration when compared with the prototypical agent fasudil. Azaindole-1 reversed hypoxic pulmonary vasoconstriction and decreased pulmonary and systemic arterial pressures in a similar manner in rats with monocrotaline-induced pulmonary hypertension. These data suggest that ROCK is involved in regulating baseline tone in the pulmonary and systemic vascular beds, and that ROCK inhibition will promote vasodilation when tone is increased by diverse stimuli including treatment with monocrotaline.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Blood Pressure; Cardiac Output; Cardiovascular System; Hypertension, Pulmonary; Hypoxia; Male; Monocrotaline; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Protein Kinase Inhibitors; Pulmonary Artery; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; Vasodilator Agents

Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung that matches blood perfusion to alveolar ventilation to optimize gas exchange. Recently we have demonstrated that acute but not sustained HPV is critically dependent on the classical transient receptor potential 6 (TRPC6) channel. However, the mechanism of TRPC6 activation during acute HPV remains elusive. We hypothesize that a diacylglycerol (DAG)-dependent activation of TRPC6 regulates acute HPV.. We investigated the effect of the DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) on normoxic vascular tone in isolated perfused and ventilated mouse lungs from TRPC6-deficient and wild-type mice. Moreover, the effects of OAG, the DAG kinase inhibitor R59949 and the phospholipase C inhibitor U73122 on the strength of HPV were investigated compared to those on non-hypoxia-induced vasoconstriction elicited by the thromboxane mimeticum U46619.. OAG increased normoxic vascular tone in lungs from wild-type mice, but not in lungs from TRPC6-deficient mice. Under conditions of repetitive hypoxic ventilation, OAG as well as R59949 dose-dependently attenuated the strength of acute HPV whereas U46619-induced vasoconstrictions were not reduced. Like OAG, R59949 mimicked HPV, since it induced a dose-dependent vasoconstriction during normoxic ventilation. In contrast, U73122, a blocker of DAG synthesis, inhibited acute HPV whereas U73343, the inactive form of U73122, had no effect on HPV.. These findings support the conclusion that the TRPC6-dependency of acute HPV is induced via DAG.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Diacylglycerol Kinase; Diglycerides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Estrenes; Hypoxia; In Vitro Techniques; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Perfusion; Piperidines; Pulmonary Circulation; Pyrrolidinones; Quinazolinones; Signal Transduction; Time Factors; TRPC Cation Channels; TRPC6 Cation Channel; Type C Phospholipases; Vasoconstriction; Vasoconstrictor Agents

Rho-kinase-dependent Ca2+ sensitization is an essential process for contraction of mammalian vascular smooth muscle but the information about its effects in non-mammalian vessels is scarce. We aimed to investigate, using the Rho-kinase inhibitor hydroxyfasudil, the potential role of the Rho-kinase pathway of Ca2+ sensitization in depolarization- and agonist-mediated contraction of chicken embryo (at day 19 of the 21 days of incubation) femoral arteries. Contraction elicited by KCl (125 mM) comprised two phases (phasic and tonic contraction), both of which were abolished in the absence of extracellular Ca2+. Hydroxyfasudil (10 microM) left the initial phasic component nearly intact but abolished the tonic component. Hydroxyfasudil also induced a marked impairment of the contractions elicited by phenylephrine (PE), the thromboxane A2 mimetic U46619, and endothelin-1. In contrast, inhibition of protein kinase C (PKC) by chelerythrine did not affect KCl- or PE-induced contractions, indicating lack of participation of PKC-mediated Ca2+ sensitization. Incubation under chronic hypoxia (15% O2 from day 0) impaired embryonic growth but did not significantly affect hydroxyfasudil-mediated relaxation. In summary, our findings are indicative of a role for Rho-kinase activity in depolarization- and agonist-induced force generation in chicken embryo femoral arteries.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Benzophenanthridines; Body Weight; Calcium; Chick Embryo; Egtazic Acid; Endothelin-1; Enzyme Inhibitors; Femoral Artery; Hypoxia; NG-Nitroarginine Methyl Ester; Oxadiazoles; Phenylephrine; Phorbol 12,13-Dibutyrate; Potassium Chloride; Protein Kinase C; Protein Kinase Inhibitors; Quinoxalines; rho-Associated Kinases; Vasoconstriction

Chronic hypoxia induces pulmonary hypertension in mice. Smooth muscle cell hyperplasia and medial thickening characterize the vasculature of these animals. Thrombospondin-1 null (TSP-1(-/-)) mice spontaneously develop pulmonary smooth muscle cell hyperplasia and medial thickening. In addition, TSP-1 produced by the pulmonary endothelium inhibits pulmonary artery smooth muscle cell growth. Based on these observations we sought to describe the pulmonary vascular changes in TSP-1(-/-) mice exposed to chronic hypoxia.. We exposed TSP-1(-/-) and wild type (WT) mice to a fraction of inspired oxygen (FiO2) of 0.1 for up to six weeks. Pulmonary vascular remodeling was evaluated using tissue morphometrics. Additionally, right ventricle systolic pressures (RVSP) and right ventricular hypertrophy by right ventricle/left ventricle + septum ratios (RV/LV+S) were measured to evaluate pulmonary hypertensive changes. Finally, acute pulmonary vasoconstriction response in both TSP-1(-/-) and WT mice was evaluated by acute hypoxia and U-46619 (a prostaglandin F2 analog) response.. In hypoxia, TSP-1(-/-) mice had significantly lower RVSP, RV/LV+S ratios and less pulmonary vascular remodeling when compared to WT mice. TSP-1(-/-) mice also had significantly lower RVSP in response to acute pulmonary vasoconstriction challenges than their WT counterparts.. TSP-1(-/-) mice had diminished pulmonary vasoconstriction response and were less responsive to hypoxia-induced pulmonary hypertension than their wild type counterparts. This observation suggests that TSP-1 could play an active role in the pathogenesis of pulmonary hypertension associated with hypoxia.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Male; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Pulmonary Artery; Thrombospondin 1; Vasoconstriction; Vasoconstrictor Agents; Ventricular Function, Right

Recent studies show that pulmonary vasodilator responses to nitrite are enhanced by hypoxia. However, the mechanism by which nitrite is converted to vasoactive nitric oxide (NO) is uncertain. In the present study, intravenous injections of sodium nitrite decreased pulmonary and systemic arterial pressures and increased cardiac output. The decreases in pulmonary arterial pressure were enhanced when tone in the pulmonary vascular bed was increased with U-46619. Under elevated tone conditions, decreases in pulmonary and systemic arterial pressures in response to nitrite were attenuated by allopurinol in a dose that did not alter responses to the NO donors, sodium nitroprusside and diethylamine/NO, suggesting that xanthine oxidoreductase is the major enzyme-reducing nitrite to NO. Ventilation with a 10% O(2) gas mixture increased pulmonary arterial pressure, and the response to hypoxia was enhanced by N(G)-nitro-l-arginine methyl ester and not altered by allopurinol. This suggests that NO formed by the endothelium and not from the reduction of plasma nitrite modulates the hypoxic pulmonary vasoconstrictor response. Although intravenous injections of sodium nitrite reversed pulmonary hypertensive responses to U-46619, hypoxia, and N(G)-nitro-l-arginine methyl ester, the pulmonary vasodilator response to nitrite was not altered by ventilation with 10% O(2) when baseline pulmonary arterial pressure was increased to similar values in animals breathing room air or the hypoxic gas. These data provide evidence that xanthine oxidoreductase is the major enzyme-reducing nitrite to vasoactive NO, and that this mechanism is not modified by hypoxia.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Allopurinol; Animals; Blood Pressure; Cardiac Output; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hydrazines; Hypoxia; Injections, Intravenous; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; Oxypurinol; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Sodium Nitrite; Time Factors; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents; Xanthine Oxidase

The small GTP-binding protein Rho and its downstream effector, Rho-kinase, are important regulators of vasoconstrictor tone. Rho-kinase is upregulated in experimental models of pulmonary hypertension, and Rho-kinase inhibitors decrease pulmonary arterial pressure in rodents with monocrotaline and chronic hypoxia-induced pulmonary hypertension. However, less is known about responses to fasudil when pulmonary vascular resistance is elevated on an acute basis by vasoconstrictor agents and ventilatory hypoxia. In the present study, intravenous injections of fasudil reversed pulmonary hypertensive responses to intravenous infusion of the thromboxane receptor agonist, U-46619 and ventilation with a 10% O(2) gas mixture and inhibited pulmonary vasoconstrictor responses to intravenous injections of angiotensin II, BAY K 8644, and U-46619 without prior exposure to agonists, which can upregulate Rho-kinase activity. The calcium channel blocker isradipine and fasudil had similar effects and in small doses had additive effects in blunting vasoconstrictor responses, suggesting parallel and series mechanisms in the lung. When pulmonary vascular resistance was increased with U-46619, fasudil produced similar decreases in pulmonary and systemic arterial pressure, whereas isradipine produced greater decreases in systemic arterial pressure. The hypoxic pressor response was enhanced by 5-10 mg/kg iv nitro-L-arginine methyl ester (L-NAME), and fasudil or isradipine reversed the pulmonary hypertensive response to hypoxia in control and in L-NAME-treated animals, suggesting that the response is mediated by Rho-kinase and L-type Ca(2+) channels. These results suggest that Rho-kinase is constitutively active in regulating baseline tone and vasoconstrictor responses in the lung under physiological conditions and that Rho-kinase inhibition attenuates pulmonary vasoconstrictor responses to agents that act by different mechanisms without prior exposure to the agonist.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Anesthesia; Angiotensin II; Animals; Blood Gas Analysis; Blood Pressure; Cardiac Output; Heart Rate; Hypoxia; Injections, Intravenous; Isradipine; Lung; Male; NG-Nitroarginine Methyl Ester; Protein Kinase Inhibitors; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; Vascular Resistance; Vasodilator Agents

Intermittent hypoxia (IH) resulting from sleep apnea can lead to pulmonary hypertension (PH) and right heart failure, similar to chronic sustained hypoxia (CH). Supplemental CO(2), however, attenuates hypoxic PH. We therefore hypothesized that, similar to CH, IH elicits PH and associated increases in arterial endothelial nitric oxide synthase (eNOS) expression, ionomycin-dependent vasodilation, and receptor-mediated pulmonary vasoconstriction. We further hypothesized that supplemental CO(2) inhibits these responses to IH. To test these hypotheses, we measured eNOS expression by Western blot in intrapulmonary arteries from CH (2 wk, 0.5 atm), hypocapnic IH (H-IH) (3 min cycles of 5% O(2)/air flush, 7 h/day, 2 wk), and eucapnic IH (E-IH) (3 min cycles of 5% O(2), 5% CO(2)/air flush, 7 h/day, 2 wk) rats and their respective controls. Furthermore, vasodilatory responses to the calcium ionophore ionomycin and vasoconstrictor responses to the thromboxane mimetic U-46619 were measured in isolated saline-perfused lungs from each group. Hematocrit, arterial wall thickness, and right ventricle-to-total ventricle weight ratios were additionally assessed as indexes of polycythemia, arterial remodeling, and PH, respectively. Consistent with our hypotheses, E-IH resulted in attenuated polycythemia, arterial remodeling, RV hypertrophy, and eNOS upregulation compared with H-IH. However, in contrast to CH, neither H-IH nor E-IH increased ionomycin-dependent vasodilation. Furthermore, H-IH and E-IH similarly augmented U-46619-induced pulmonary vasoconstriction but to a lesser degree than CH. We conclude that maintenance of eucapnia decreases IH-induced PH and upregulation of arterial eNOS. In contrast, increases in pulmonary vasoconstrictor reactivity following H-IH are unaltered by exposure to supplemental CO(2).

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Carbon Dioxide; Chronic Disease; Disease Models, Animal; Dose-Response Relationship, Drug; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypocapnia; Hypoxia; Ionomycin; Ionophores; Male; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxygen; Polycythemia; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

Intravital microscopy (IVM) is considered as the gold standard for in vivo investigations of dynamic microvascular regulation. The availability of transgenic and knockout animals has propelled the development of murine IVM models for various organs, but technical approaches to the pulmonary microcirculation are still scarce. In anesthetized and ventilated BALB/c mice, we established a microscopic access to the surface of the right upper lung lobe by surgical excision of a window of 7- to 10-mm diameter from the right thoracic wall. The window was covered by a transparent polyvinylidene membrane and sealed with alpha-cyanoacrylate. Removal of intrathoracic air via a trans-diaphragmal intrapleural catheter coupled the lung surface to the window membrane. IVM preparations were hemodynamically stable for at least 120 min, with mean arterial blood pressure above 70 mmHg, and mean arterial Po(2) and arterial Pco(2) in the range of 90-100 Torr and 30-40 Torr, respectively. Imaged lungs did not show any signs of acute lung injury or edema. Following infusion of FITC dextran, subpleural pulmonary arterioles and venules of up to 50-microm diameter and alveolar capillary networks could be visualized during successive expiratory plateau phases over a period of at least 2 h. Vasoconstrictive responses to hypoxia (11% O(2)) or infusion of the thromboxane analog U-46619 were prominent in medium-sized arterioles (30- to 50-microm diameter), minor in small arterioles <30 microm, and absent in venules. The presented IVM model may constitute a powerful new tool for investigations of pulmonary microvascular responses in mice.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arterioles; Blood Pressure; Capillaries; Dextrans; Exhalation; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Hypoxia; Lung; Male; Mice; Mice, Inbred BALB C; Microscopy, Video; Models, Animal; Pulmonary Circulation; Time Factors; Vasoconstriction; Vasoconstrictor Agents; Venules

Endothelin-1 (ET-1), a potent vasoconstrictor, is believed to contribute to the pathogenesis of hypoxic pulmonary hypertension. Previously we demonstrated that contraction induced by ET-1 in intrapulmonary arteries (IPA) from chronically hypoxic (CH) rats occurred independently of changes in intracellular Ca2+ concentration ([Ca2+]i), suggesting that ET-1 increased Ca2+ sensitivity. The mechanisms underlying this effect are unclear but could involve the activation of myosin light chain kinase, Rho kinase, PKC, or tyrosine kinases (TKs), including those from the Src family. In this study, we examined the effect of pharmacological inhibitors of these kinases on maximum tension generated by IPA from CH rats (10% O2 for 21 days) in response to ET-1. Experiments were conducted in the presence of nifedipine, an L-type Ca2+ channel blocker, to isolate the component of contraction that occurred without a change in [Ca2+]i. The mean change in tension caused by ET-1 (10(-8) M) expressed as a percent of the maximum response to KCl was 184.0+/-39.0%. This response was markedly inhibited by the Rho kinase inhibitors Y-27632 and HA-1077 and the TK inhibitors genistein, tyrphostin A23, and PP2. In contrast, staurosporine and GF-109203X, inhibitors of PKC, had no significant inhibitory effect on the tension generated in response to ET-1. We conclude that the component of ET-1-induced contraction that occurs without a change in [Ca2+]i in IPA from CH rats requires activation of Rho kinase and TKs, but not PKC.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Amides; Animals; Azepines; Endothelin-1; Enzyme Inhibitors; Genistein; Hypoxia; Indoles; Intracellular Signaling Peptides and Proteins; Isometric Contraction; Male; Maleimides; Myosin-Light-Chain Kinase; Potassium Chloride; Protein Kinase C; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Pulmonary Artery; Pyridines; Pyrimidines; Rats; Rats, Wistar; rho-Associated Kinases; src-Family Kinases; Staurosporine; Vasoconstriction

We have previously shown that pentose phosphate pathway (PPP) inhibitors, 6-aminonicotinamide (6-AN) and epiandrosterone (EPI), markedly reduce hypoxic pulmonary vasoconstriction (HPV). Although it has been suggested that changes in the NADPH/NADP+ ratio and redox status are involved in the mechanism of HPV, the role of PPP-derived NADPH in this phenomenon is not known. The aim of this study, therefore, was to investigate the role of PPP-derived NADPH in HPV using isolated rat pulmonary arteries (PA) and perfused rat lungs. The NADPH/NADP+ ratio and NADPH levels in PA and lungs exposed to hypoxia increased 2-fold and 7-fold, respectively, compared to time-matched normoxic controls. Both hypoxia-induced increases in lung NADPH levels and lung perfusion pressure were inhibited by 6-AN (500 microM) or EPI (300 microM). The chemical inhibitors of PPP and hypoxia similarly decreased lung tissue NOx levels by approximately 50%. In contrast, hypoxia increased the lung soluble guanylate cyclase (sGC) activity (from 22.9+/-6.3 to 57.1+/-7.6 pmol/min/g), which was prevented by PPP inhibitors. ODQ, a sGC inhibitor, potentiated HPV. These results suggest that while PPP-derived NADPH may play a significant role in HPV, it may also moderate the magnitude of HPV through activation of the NO-sGC-cGMP vasodilation pathway.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 6-Aminonicotinamide; Androsterone; Animals; Enzyme Inhibitors; Glucose-6-Phosphate; Glucosephosphate Dehydrogenase; Guanylate Cyclase; Hypoxia; In Vitro Techniques; Lung; Male; NADP; Nitrates; Nitrites; Oxadiazoles; Pentose Phosphate Pathway; Perfusion; Pulmonary Artery; Quinoxalines; Rats; Rats, Sprague-Dawley; Vascular Resistance; Vasoconstriction

PPHN, caused by perinatal hypoxia or inflammation, is characterized by an increased thromboxane-prostacyclin ratio and pulmonary vasoconstriction. We examined effects of hypoxia on myocyte thromboxane responsiveness. Myocytes from 3rd-6th generation pulmonary arteries of newborn piglets were grown to confluence and synchronized in contractile phenotype by serum deprivation. On the final 3 days of culture, myocytes were exposed to 10% O2 for 3 days; control myocytes from normoxic piglets were cultured in 21% O2. PPHN was induced in newborn piglets by 3-day hypoxic exposure (Fi(O2) 0.10); pulmonary arterial myocytes from these animals were maintained in normoxia. Ca2+ mobilization to thromboxane mimetic U-46619 and ATP was quantified using fura-2 AM. Three-day hypoxic exposure in vitro results in increased basal [Ca2+]i, faster and heightened peak Ca2+ response, and decreased U-46619 EC50. These functional changes persist in myocytes exposed to hypoxia in vivo but cultured in 21% O2. Blockade of Ca2+ entry and store refilling do not alter peak U-46619 Ca2+ responses in hypoxic or normoxic myocytes. Blockade of ryanodine-sensitive or IP3-gated intracellular Ca2+ channels inhibits hypoxic augmentation of peak U-46619 response. Ca2+ response to ryanodine alone is undetectable; ATP-induced Ca2+ mobilization is unaltered by hypoxia, suggesting no independent increase in ryanodine-sensitive or IP3-linked intracellular Ca2+ pool mobilization. We conclude hypoxia has a priming effect on neonatal pulmonary arterial myocytes, resulting in increased resting Ca2+, thromboxane hypersensitivity, and hyperreactivity. We postulate that hypoxia increases agonist-induced TP-R-linked IP3 pathway activation. Myocyte thromboxane hyperresponsiveness persists in culture after removal from the initiating hypoxic stimulus, suggesting altered gene expression.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Animals, Newborn; Calcium; Calcium Channel Blockers; Cells, Cultured; Disease Models, Animal; Heart Ventricles; Humans; Hypoxia; Infant, Newborn; Macrocyclic Compounds; Muscle Cells; Nifedipine; Organ Size; Oxazoles; Persistent Fetal Circulation Syndrome; Pulmonary Artery; Receptors, Thromboxane; Ryanodine; Swine; Vasoconstriction

Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion with ventilation to optimize pulmonary gas exchange. However, it remains unclear whether acute HPV (occurring within seconds) and the vasoconstrictor response to sustained alveolar hypoxia (developing over several hours) are triggered by identical mechanisms. We investigated the effect of mitochondrial and NADPH oxidase inhibitors on both phases of HPV in intact rabbit lungs. These studies revealed that the sustained HPV is largely dependent on mitochondrial complex I and totally dependent on complex IV, whereas NADPH oxidase dependence was only observed for acute HPV. These findings were reinforced by an alternative approach employing lungs from mice deficient in the NADPH oxidase subunit p 47(phox). In these mice (which lack a subunit suggested to be important for the function of most NADPH oxidase isoforms), but not in gp 91(phox)-deficient mice (which represent only one isoform of NADPH oxidases), acute HPV was significantly reduced, while non-hypoxia-induced vasoconstrictions elicited by the thromboxane mimetic U46619 were not affected. We concluded that the acute phase and the sustained phase of HPV are differentially regulated, with NADPH oxidase activity predominating in the acute phase, while a strong dependence on mitochondrial participation was observed for the second phase.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Electron Transport Complex I; Electron Transport Complex II; Electron Transport Complex III; Electron Transport Complex IV; Female; Hypoxia; In Vitro Techniques; Lung; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mitochondria; NADPH Oxidase 2; NADPH Oxidases; Oxygen; Protein Isoforms; Pulmonary Gas Exchange; Rabbits; Superoxides; Vasoconstriction; Vasoconstrictor Agents

The ability of the smooth muscle of the human umbilical artery to relax may vary under physiological and pathological conditions. We investigated the responsiveness of that preparation to relaxation, as well as the influence of reduced oxygen condition on these responses. Rings of human umbilical arteries from full-term Caesarian deliveries were suspended in modified Krebs-Henseleit solutions bubbled with a gas mixture of 95% O2:5% CO2 (normal oxygen condition) or 2.5% O2:8% CO2 balanced with N2 (reduced oxygen condition). These rings were contracted with potassium chloride, serotonin or the thromboxane A2 mimetic U46619, before being exposed to either the nitrovasodilator sodium nitroprusside, the potassium channel opener levcromakalim or the calcium channel antagonist amlodipine. While sodium nitroprusside elicited relaxation in this blood vessel, the maximal relaxation to the nitric oxide donor was significantly smaller than that induced by levcromakalim and amlodipine. The nature of the constrictor agent used, or changes of oxygen conditions did not significantly affect the relaxation profile of this human blood vessel. These data suggest that the smooth muscle of the human umbilical artery may be less responsive to vasodilators that act via the nitric oxide pathway. Moreover, vascular responses of umbilical arterial smooth muscle to relaxing agents do not alter under hypoxic or different vasoconstricting conditions.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Amlodipine; Cromakalim; Humans; Hypoxia; Muscle, Smooth, Vascular; Nitric Oxide; Nitroprusside; Potassium Chloride; Serotonin; Thromboxane A2; Umbilical Arteries; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

Vascular endothelial growth factor (VEGF) is a potent mitogen with angiogenic and vasoactive properties. VEGF can bind to two types of receptors. VEGF receptor 2 (VEGFR2) is mainly responsible for the dilator response to VEGF through nitric oxide (NO) release, whereas VEGFR1 may sequestrate the ligand. We hypothesized that in neonatal hypoxia-induced pulmonary hypertension, VEGF vasodilation is reduced. The dilator response to VEGF was assessed in isolated perfused lung of 1-d-old piglets that were exposed to either normoxia or hypoxia (fraction of inspired oxygen 0.10) for 14 d. The plasma and pulmonary artery concentration of VEGF was measured by quantitative sandwich enzyme immunoassay in piglets that were exposed to either normoxia or hypoxia for 1, 3, 7, or 14 d. The expression of VEGFR1, VEGFR2, and endothelial NO synthase in pulmonary artery was measured in the same study groups using Western blot analysis. VEGF (10(-12)-10(-9) M) induces a dose-dependent relaxation in 14-d normoxic piglets, whereas vasodilation is abolished after 14 d of hypoxia. VEGF tissue concentration is increased by hypoxia. VEGFR1 expression is dramatically increased after 1, 3, and 7 d of hypoxia compared with normoxia and returns to normal afterward. VEGFR2 expression is reduced by hypoxia at 14 d. However, endothelial NO synthase expression is not affected by hypoxia compared with normoxia. In neonatal hypoxia-induced pulmonary hypertension, VEGF is increased, whereas vasodilation to VEGF is abolished. This reduced vasodilation may be due to decreased VEGFR2 expression. We speculate that sequestration by VEGFR1 may also limit, to some extent, the vascular protecting effect of VEGF, thus contributing to the pathophysiologic changes seen in neonatal hypoxia-induced pulmonary hypertension.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Animals, Newborn; Blotting, Western; Cytosol; Endothelium, Vascular; Hypertension, Pulmonary; Hypoxia; Ligands; Lung; Muscle, Smooth; Nitric Oxide Synthase; Oxygen; Perfusion; Swine; Time Factors; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2; Vasoconstrictor Agents

Acute alveolar hypoxia causes pulmonary vasoconstriction that matches lung perfusion to ventilation to optimize gas exchange. Chronic alveolar hypoxia induces pulmonary hypertension, characterized by increased muscularization of the pulmonary vasculature and right ventricular hypertrophy. Elevated erythropoietin (EPO) plasma levels increase hematocrit and blood viscosity and may affect structure and function of the pulmonary circulation. To differentiate between the direct effects of hypoxia and those linked to a hypoxia-induced increase in EPO/hematocrit levels, we investigated the lung vasculature in transgenic mice constitutively over-expressing EPO (termed tg6) upon exposure to normoxia and chronic hypoxia. Despite increased hematocrit levels (approximately 0.86),tg6 mice kept in normoxia did not develop selective right ventricular hypertrophy. The portion of vessels with a diameter of 51-95 microm and >155 microm was increased whereas the portion of small vessels (30-50 microm) was decreased. Pulmonary vascular resistance and the strength of hypoxic vasoconstriction measured in isolated perfused lungs were decreased. Vasoconstrictions induced by the thromboxane mimetic U46619 tended to be reduced. After chronic hypoxia (FiO2 = 0.10, 21 days), vascular resistance and vasoconstrictor responses to acute hypoxia and U46619 were reduced in tg6 mice compared to wildtype controls. Chronic hypoxia increased the degree of pulmonary vascular muscularization in wildtype but not in tg6 mice that already exhibited less muscularization in normoxia. In conclusion, congenital over-expression of EPO exerts an "anti-pulmonary hypertensive" effect, both structurally and functionally, particularly obvious upon chronic hypoxia.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aspirin; Blood Pressure; Blood Vessels; Blood Viscosity; Cyclooxygenase Inhibitors; Erythropoietin; Hematocrit; Hypertension, Pulmonary; Hypertrophy, Left Ventricular; Hypertrophy, Right Ventricular; Hypoxia; Lung; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muscle, Smooth, Vascular; Nitric Oxide Synthase; omega-N-Methylarginine; Up-Regulation; Vascular Resistance; Vasoconstriction; Vasoconstrictor Agents

1. The purpose of this study was to investigate whether a membrane-permeable superoxide dismutase mimetic, tempol, added either alone or in combination with the nitric oxide (NO) donor molsidomine, prevents the development of pulmonary hypertension (PH) in chronic hypoxic rats. 2. Chronic hypobaric hypoxia (10% oxygen) for 2 weeks increased the right ventricular systolic pressure (RVSP), right ventricle and lung wet weight. Relaxations evoked by acetylcholine (ACh) and the molsidomine metabolite SIN-1 were impaired in isolated proximal, but not distal pulmonary arteries, from chronic hypoxic rats. 3. Treatment with tempol (86 mg x kg(-1) day(-1) in drinking water) normalized RVSP and reduced right ventricular hypertrophy, while systemic blood pressure, lung and liver weights, and blunted ACh relaxation of pulmonary arteries were unchanged. 4. Treatment with molsidomine (15 mg x kg(-1) day(-1) in drinking water) had the same effects as tempol, except that liver weight was reduced, and potassium and U46619-evoked vasoconstrictions in pulmonary arteries were increased. Combining tempol and molsidomine did not have additional effects compared to tempol alone. ACh relaxation in pulmonary arteries was not normalized by these treatments. 5. The media to lumen diameter ratio of the pulmonary arteries was greater for the hypoxic rats compared to the normoxic rats, and was not reversed by treatment with tempol, molsidomine, or the combination of tempol and molsidomine. 6. We conclude that tempol, like molsidomine, is able to correct RVSP and reduce right ventricular weight in the rat hypoxic model. Functional and structural properties of pulmonary small arteries were little affected. The results support the possibility that superoxide dismutase mimetics may be a useful means for the treatment of PH.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetylcholine; Administration, Oral; Animals; Body Weight; Chronic Disease; Cyclic N-Oxides; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Synergism; Drug Therapy, Combination; Endothelin-1; Free Radical Scavengers; Heart Rate; Hypertrophy, Right Ventricular; Hypoxia; Male; Molsidomine; Muscle, Smooth, Vascular; Organ Size; Pulmonary Artery; Rats; Rats, Wistar; Spin Labels; Superoxide Dismutase; Vasoconstriction; Vasodilation; Ventricular Pressure

Pulmonary vascular remodeling during chronic hypoxia may be the result of either oxygen deprivation or erythrocytosis. To separate experimentally the effects of hypoxia and erythrocytosis, we analyzed transgenic mice that constitutively overexpress the human erythropoietin gene in an oxygen-independent manner. These mice are characterized by polycythemia but have normal blood pressure, heart rate, and cardiac output. In transgenic mice, pulmonary artery pressure (PAP) was increased in vivo but was reduced in blood-free perfused lungs. The thromboxane receptor agonist U46619 caused a smaller rise in PAP in isolated transgenic lungs than in lungs from wild-type mice. The transgenic pulmonary vasculature was characterized by elevated prostacyclin production, stronger endothelial nitric oxide synthase expression, and reduced pulmonary vascular smooth muscle thickness. The fact that transgenic polycythemic mice have marked pulmonary hypertension in vivo but not in vitro suggests that their pulmonary hypertension is due to the increased blood viscosity, thus supporting an independent role of polycythemia in the development of pulmonary hypertension. In addition, our findings indicate that the lungs of transgenic animals adapt to the high PAP by elevated synthesis of vasodilators and reduced vascular smooth muscle thickness that tend to reduce vascular tone and vascular responsiveness.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Analysis of Variance; Animals; Blood Viscosity; Erythropoietin; Hypertension, Pulmonary; Hypoxia; Immunohistochemistry; Lung; Mice; Mice, Transgenic; Muscle, Smooth, Vascular; Polycythemia; Pulmonary Artery; Vasoconstrictor Agents

We have shown that the specific inhibition of hypoxia-induced relaxation by organ culture in porcine coronary arteries can be mimicked by treatment of control vessels with the protein synthesis inhibitor, cycloheximide. We hypothesize that organ culture of vascular smooth muscle results in the decreased expression of proteins that are critical for vascular oxygen sensing. Using two-dimensional gel electrophoresis and mass spectroscopy, we identified such candidate proteins. The expressions of the smooth muscle-specific protein, SM22, and tropomyosin are decreased after 24 h in organ culture. These results were confirmed by Western blot analysis. Other smooth muscle proteins (actin and calponin) exhibited little change. We also demonstrate a 50% downregulation in the small G protein, Rho, a potent modulator of Ca(2+)-independent force. These results indicate that organ culture preferentially inhibits the expression of certain smooth muscle proteins. This change in protein expression after organ culture correlates with the specific inhibition of hypoxic vasorelaxation. These results provide novel target pathways for investigation that are potentially important for vascular oxygen sensing.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Bridged Bicyclo Compounds, Heterocyclic; Coronary Vessels; Down-Regulation; Electrophoresis, Gel, Two-Dimensional; Fatty Acids, Unsaturated; Hydrazines; Hypoxia; Microfilament Proteins; Muscle Proteins; Muscle, Smooth, Vascular; Organ Culture Techniques; Oxygen; Proteomics; rho GTP-Binding Proteins; Swine; Tropomyosin; Vasoconstrictor Agents

Previous work suggests that superoxide mediates hypoxia/reoxygenation (H/R)-induced constriction of isolated mouse coronary arteries (CA). To determine the source of superoxide overproduction during H/R we studied CA obtained from transgenic (Tg) mice overexpressing human CuZn-superoxide dismutase (SOD) and mice lacking gp91(phox) using an in vitro vascular ring bioassay. We found that under normoxic conditions CA isolated from wild type (wt) mice, CuZn-SOD Tg mice and gp91(phox) knock-out mice had similar contractile responses to U46619 and hypoxia and similar dilation responses to acetylcholine. In wt CA, 30 min of hypoxia (1% O(2)) followed by reoxygenation (16% O(2)) resulted in further coronary vasoconstriction (internal diameter from 105 +/- 11 to 84.5 +/- 17.9 microm), whereas this response was completely blocked in both CuZn-SOD Tg and gp91(phox) knock-out CA (104.3 +/- 10.5 to 120.7 +/- 14 microm and 143.3 +/- 15.3 to 172.7 +/- 12.5 microm, respectively, p < 0.01). Furthermore, we show that H/R enhances the generation of superoxide radicals in wt CA (25.8 +/- 0.7 relative light units per second (RLU/s)), whereas CuZn-SOD Tg CA (12.2 +/- 0.8 RLU/s, p < 0.01) and gp91(phox) CA (12.5 +/- 0.9 RLU/s, p < 0.01) show reduced levels. These results demonstrate that H/R-induced vasoconstriction is mediated by intracellular superoxide overproduction via endothelial NADPH oxidase gp91(phox). Therefore, increasing endogenous levels of CuZn-SOD in CA may provide a novel cardioprotective strategy for maintaining coronary perfusion under conditions of H/R.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetylcholine; Animals; Arteries; Biological Assay; Coronary Vessels; Endothelium, Vascular; Humans; Hypoxia; Luminescent Measurements; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Models, Biological; NADPH Oxidase 2; NADPH Oxidases; Oxygen; RNA, Messenger; Superoxide Dismutase; Superoxides; Time Factors; Vasoconstriction

Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion with ventilation which tends to optimize pulmonary gas exchange. Investigations using genetically engineered mice represent a promising approach to understand the underlying mechanisms. Our goal was to characterize basic features of HPV in the isolated buffer-perfused and ventilated mouse lung system. HPV was reproducible for several hours when ventilating the lungs with 1% O2 (10 min) alternated with normoxic ventilation periods (21% O2, 15 min). HPV was well elicitable and most constant using Krebs-Henseleit buffer with the addition of hydroxyethylamylopectin as an oncotic agent. Inhibition of both lung NO and prostanoid formation amplified HPV in an over-additive fashion. HPV was higher in BALB/c mive as compared to C57BL/6 mice, and was approximately threefold enhanced under positive pressure ventilation as compared to negative pressure ventilation. A three hour hypoxic ventilation period resulted in a biphasic vasoconstrictor response with loss of posthypoxic vasodilatation. In summary, we have characterised HPV and established an experimental set-up optimized for investigation of the basic mechanisms of HPV in mice.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Amylopectin; Animals; Aspirin; Blood Pressure; Drug Interactions; Enzyme Inhibitors; Hypoxia; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; omega-N-Methylarginine; Oxygen; Pulmonary Artery; Pulmonary Circulation; Pulmonary Gas Exchange; Species Specificity; Tidal Volume; Vasoconstriction; Vasoconstrictor Agents

The role of S-nitrosated hemoglobin (SNO-Hb) in the regulation of blood flow is a central and controversial question in cardiopulmonary physiology. In the present study, we investigate whether intact human red blood cells (RBCs) synthesized to contain high SNO-Hb levels are able to export nitric oxide bioactivity and vasodilate the pulmonary circulation, and whether SNO-Hb dependent vasodilation occurs secondary to an intrinsic oxygen-linked, allosteric function of Hb. RBCs containing supraphysiological concentrations (100-1,000x normal) of SNO-Hb (SNO-RBCs) were synthesized and added to isolated, perfused rat lungs during anoxic or normoxic ventilation, and during normoxic ventilation with pulmonary hypertension induced by the thromboxane mimetic U-46619. SNO-RBCs produced dose-dependent pulmonary vasodilation compared with control RBCs during conditions of both normoxic (U-46619) and hypoxic pulmonary vasoconstriction. These effects were associated with a simultaneous, rapid, and temperature-dependent loss of SNO from Hb. Both vasodilatory effects and the rate of SNO-Hb degradation were independent of oxygen tension and Hb oxygen saturation. Furthermore, these effects were not affected by inhibition of the RBC membrane band 3 protein (anion exchanger-1), a putative membrane facilitator of NO export from RBCs. Whereas these data support observations by multiple groups that synthesized SNO-Hb can vasodilate, this effect is not under intrinsic oxygen-dependent allosteric control, nor likely to be relevant in the pulmonary circulation at normal physiological concentrations.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Anion Exchange Protein 1, Erythrocyte; Erythrocytes; Hemoglobins; Humans; Hypoxia; Nitric Oxide; Nitrosation; Oxygen; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Vasoconstriction; Vasoconstrictor Agents; Vasodilation

The blood anion nitrite contributes to hypoxic vasodilation through a heme-based, nitric oxide (NO)-generating reaction with deoxyhemoglobin and potentially other heme proteins. We hypothesized that this biochemical reaction could be harnessed for the treatment of neonatal pulmonary hypertension, an NO-deficient state characterized by pulmonary vasoconstriction, right-to-left shunt pathophysiology and systemic hypoxemia. To test this, we delivered inhaled sodium nitrite by aerosol to newborn lambs with hypoxic and normoxic pulmonary hypertension. Inhaled nitrite elicited a rapid and sustained reduction ( approximately 65%) in hypoxia-induced pulmonary hypertension, with a magnitude approaching that of the effects of 20 p.p.m. NO gas inhalation. This reduction was associated with the immediate appearance of NO in expiratory gas. Pulmonary vasodilation elicited by aerosolized nitrite was deoxyhemoglobin- and pH-dependent and was associated with increased blood levels of iron-nitrosyl-hemoglobin. Notably, from a therapeutic standpoint, short-term delivery of nitrite dissolved in saline through nebulization produced selective, sustained pulmonary vasodilation with no clinically significant increase in blood methemoglobin levels. These data support the concept that nitrite is a vasodilator acting through conversion to NO, a process coupled to hemoglobin deoxygenation and protonation, and evince a new, simple and inexpensive potential therapy for neonatal pulmonary hypertension.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Administration, Inhalation; Aerosols; Animals; Animals, Newborn; Blood Pressure; Cardiac Output; Disease Models, Animal; Hemoglobins; Humans; Hydrogen-Ion Concentration; Hypoxia; Infant, Newborn; Methemoglobin; Nitric Oxide; Oxygen; Persistent Fetal Circulation Syndrome; Sheep; Sodium Nitrite; Vasodilator Agents

This study further investigates the mechanisms responsible for the effects of acute and severe hypoxia, and subsequent reoxygenation, on the contractility of isolated rat mesenteric arteries. In noradrenaline (NA)-contracted arteries, hypoxia caused a relaxation to near baseline levels. Reoxygenation resulted in an immediate transient contraction before tension returned more slowly to prehypoxia levels. Similar responses to hypoxia were observed in tissues precontracted by addition of KCl (60 mM) or U46619 (10 microM); however, the transient contraction upon reoxygenation was absent (KCl) or reduced (U46619). Responses to hypoxia were independent of changes in intracellular calcium ([Ca2+]i), while those to reoxygenation were accompanied by corresponding changes in [Ca2+]i and were completely abolished by ryanodine. In NA-contracted tissues, all responses were unaffected by endothelial removal or by inhibitors of nitric oxide synthase and cyclooxygenase. The K+ channel blockers triethylamine (TEA), glibenclamide, and 4-aminopyridine (4-AP) had no effect on the responses to hypoxia. The transient contractile response to reoxygenation was, however, significantly reduced in the presence of 4-AP. The response to reoxygenation, but not that to hypoxia, was inhibited by the antioxidant dithiothreitol (DTT) and the NAD(P)H-oxidase inhibitor diphenyliodonium (DPI). These data suggest that hypoxic vasodilation occurs independently of reductions in [Ca2+]i. Alternatively, transient contractions on reoxygenation are dependent upon the generation of reactive oxygen species and the release of stored Ca2+.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 4-Aminopyridine; Adenosine Triphosphate; Animals; Anti-Arrhythmia Agents; Calcium; Endothelium, Vascular; Ethylamines; Glyburide; Hypoxia; Male; Mesenteric Arteries; Muscle Contraction; Norepinephrine; Oxygen; Potassium; Potassium Channel Blockers; Potassium Channels; Rats; Rats, Wistar; Reperfusion Injury; Ryanodine

Chronic hypoxia causes redistribution of fetal cardiac output by mechanisms poorly understood. We tested the hypothesis that chronic hypoxia alters vascular reactivity of arteries from near-term fetal guinea pigs.. Pregnant guinea pigs (50 days, term = 65 days) were exposed to either normoxia (room air) or hypoxia (12% O2) for 14 days. Carotid artery ring segments from anesthetized fetuses were mounted onto myographs for measurement of force. Contractile responses to cumulative addition of prostaglandin F2alpha (PGF2alpha, 10(-9) M to 10(-5) M), U46619, a thromboxane mimetic (10(-12) M to 12(-6) M), and KCl (10 to 120 mM) were measured in the presence and absence of INDO (INDO, 10(-5) M) alone and INDO plus nitro-L-arginine (LNA, 10(-4) M), or INDO plus N6-iminoethyl-L-lysine (LNIL, 5 x 10(-5) M, a selective iNOS inhibitor), and measured in endothelium-intact and denuded arteries. Nitric oxide synthase (NOS) activity was measured in isolated arteries by 14C-L-arginine to 14C-L-citrulline conversion.. Hypoxia decreased contractile responses to both PGF2alpha and U46619 under control conditions. Maximal contraction to both agonists was increased in hypoxemic arteries after INDO alone and INDO + LNA compared to normoxic controls. Endothelium-denudation abolished the differences between the groups. KCl contraction was unaffected by hypoxia. LNIL potentiated maximal PGF(2alpha) contraction but was similar between groups. Hypoxia increased (P < .05) total and Ca(2+)-dependent NOS activities by 1.7- and 2.1-fold, respectively, but had no effect on Ca(2+)-independent activity.. Chronic hypoxia alters vascular reactivity of fetal carotid arteries by increasing the contribution of both vasodilator prostaglandins and nitric oxide and suggests that changes in local vascular mechanisms may be altered by chronic hypoxia.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arteries; Calcium; Carotid Arteries; Dinoprost; Endothelium, Vascular; Female; Fetal Weight; Guinea Pigs; Hypoxia; Muscle Contraction; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Potassium Chloride; Pregnancy; Prostaglandins; Vasoconstrictor Agents

Our purpose was to determine whether production of arachidonic acid metabolites, particularly cyclooxygenase (COX) metabolites, is altered in 100-400-microm-diameter pulmonary arteries of piglets at an early stage of pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for 3 days. A cannulated artery technique was used to measure responses of 100-400-microm-diameter pulmonary arteries to arachidonic acid, a prostacyclin analog, or the thromboxane mimetic. Radioimmunoassay was used to determine pulmonary artery production of thromboxane B(2) (TxB(2)) and 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)), the stable metabolites of thromboxane and prostacyclin, respectively. Assessment of abundances of COX pathway enzymes in pulmonary arteries was determined by immunoblot technique. Arachidonic acid induced less dilation in pulmonary arteries from hypoxic than in pulmonary arteries from control piglets. Pulmonary artery responses to prostacyclin and were similar for both groups. 6-Keto-PGF(1alpha) production was reduced, whereas TxB(2) production was increased in pulmonary arteries from hypoxic piglets. Abundances of both COX-1 and prostacyclin synthase were reduced, whereas abundances of both COX-2 and thromboxane synthase were unaltered in pulmonary arteries from hypoxic piglets. At least partly due to altered abundances of COX pathway enzymes, a shift in production of arachidonic acid metabolites, away from dilators toward constrictors, may contribute to the early phase of chronic hypoxia-induced pulmonary hypertension in newborn piglets.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 6-Ketoprostaglandin F1 alpha; Animals; Animals, Newborn; Arachidonic Acid; Chronic Disease; Cyclooxygenase 1; Cyclooxygenase 2; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Hypertension, Pulmonary; Hypoxia; Intramolecular Oxidoreductases; Isoenzymes; Prostaglandin-Endoperoxide Synthases; Pulmonary Artery; Reference Values; Swine; Thromboxane B2; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion to ventilation for optimizing pulmonary gas exchange. Chronic alveolar hypoxia results in vascular remodeling and pulmonary hypertension. Previous studies have reported conflicting results of the effect of chronic alveolar hypoxia on pulmonary vasoreactivity and the contribution of nitric oxide (NO), which may be related to species and strain differences as well as to the duration of chronic hypoxia. Therefore, we investigated the impact of chronic hypoxia on HPV in rabbits, with a focus on lung NO synthesis. After exposure of the animals to normobaric hypoxia (10% O(2)) for 1 day to 10 wk, vascular reactivity was investigated in ex vivo perfused normoxic ventilated lungs. Chronic hypoxia induced right heart hypertrophy and increased normoxic vascular tone within weeks. The vasoconstrictor response to an acute hypoxic challenge was strongly downregulated within 5 days, whereas the vasoconstrictor response to the thromboxane mimetic U-46619 was maintained. The rapid downregulation of HPV was apparently not linked to changes in the lung vascular NO system, detectable in the exhaled gas and by pharmacological blockage of NO synthesis. Treatment of the animals with long-term inhaled NO reduced right heart hypertrophy and partially maintained the reactivity to acute hypoxia, without any impact on the endogenous NO system being noted. We conclude that chronic hypoxia causes rapid downregulation of acute HPV as a specific event, preceding the development of major pulmonary hypertension and being independent of the lung vascular NO system. Long-term NO inhalation partially maintains the strength of the hypoxic vasoconstrictor response.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acute Disease; Animals; Breath Tests; Chronic Disease; Disease Models, Animal; Enzyme Inhibitors; Female; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; In Vitro Techniques; Lung; Male; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Rabbits; Time; Vasoconstriction; Vasoconstrictor Agents

We previously found that nitric oxide synthase (NOS) inhibition fully blocked alkalosis-induced relaxation of piglet pulmonary artery and vein rings. In contrast, NOS inhibition alone had no effect on alkalosis-induced pulmonary vasodilation in isolated piglet lungs. This study sought to identify factors contributing to the discordance between isolated and in situ pulmonary vessels. The roles of pressor stimulus (hypoxia vs. the thromboxane mimetic U-46619), perfusate composition (blood vs. physiological salt solution), and flow were assessed. Effects of NOS inhibition on alkalosis-induced dilation were also directly compared in 150-350-microm-diameter cannulated arteries and 150-900-microm-diameter, angiographically visualized, in situ arteries. Finally, effects of NOS inhibition on alkalosis-induced vasodilation were measured in intact piglets. NOS inhibition with N(omega)-nitro-L-arginine fully abolished alkalosis-induced vasodilation in all cannulated arteries but failed to alter alkalosis-induced vasodilation in intact lungs. The results indicate that investigation of other factors, such as perivascular tissue (e.g., adventitia and parenchyma) and remote signaling pathways, will need to be carried out to reconcile this discordance between isolated and in situ arteries.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Alkalosis; Animals; Animals, Newborn; Blood Flow Velocity; Catheterization; Enzyme Inhibitors; Hypoxia; Nitroarginine; Perfusion; Pulmonary Artery; Pulmonary Circulation; Swine; Vasoconstrictor Agents; Vasodilation

Relaxation induced by bradykinin is diminished by hypoxia in epicardial coronary arteries. The bradykinin-degrading enzyme, neutral endopeptidase (NEP, EC.3.4.24.11), is a potential target for coronary artery vasodilators. In this study, we examined the effect of thiorphan, an inhibitor of NEP, on the tone of porcine isolated coronary artery under hypoxic conditions. Endothelium-intact porcine isolated coronary artery rings were isometrically contracted with a prostaglandin F(2alpha) analogue (U46619, 0.75 microM) and potassium chloride (KCl, 30 mM), and relaxed with bradykinin (1-1000 nM) under normoxic (partial pressure of oxygen, pO(2) approximately 90-100 mmHg) and moderately hypoxic (pO(2) approximately 50-60 mmHg) conditions. Experiments were performed to study the effects of 30 min pre-treatment with the NEP-inhibitor, thiorphan (10 microM), both at physiological and at low pO(2)s. Hypoxia inhibited the bradykinin-induced relaxation in porcine epicardial coronary arteries. In normoxia, thiorphan significantly enhanced the decrease of coronary tone produced by bradykinin (1-10 nM) when U46619 was used as contractile agent. Under hypoxic conditions, in U46619 contracture, thiorphan did not influence, but in KCl contracture it enhanced the magnitude of relaxations induced by bradykinin. In the absence of bradykinin, thiorphan had no significant effect on the basal, KCl- and U46619-elevated tones and on the hypoxia-induced decrease of coronary artery tone. Inhibition of NEP-enzyme activity may effectively improve the relaxing capacity of epicardial coronary arteries under hypoxic/hyperkalemic conditions. This effect could be potentially utilized when the endothelial function and relaxation of the coronary arteries are impaired under clinical conditions.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Bradykinin; Coronary Vessels; Drug Synergism; Hyperkalemia; Hypoxia; In Vitro Techniques; Muscle Relaxation; Muscle, Smooth, Vascular; Potassium Chloride; Protease Inhibitors; Swine; Thiorphan; Vasoconstrictor Agents; Vasodilator Agents

Tyrosine kinase pathway has been shown to be involved in the effects of hypoxia in pulmonary arteries, but its role in pulmonary vein is not known. The aims of this study were to determine the effect of hypoxia in sheep isolated pulmonary veins and to identify the role of tyrosine kinase pathway in hypoxic response. Genistein and tyrphostin were used as selective tyrosine kinase inhibitors, and sodium orthovanadate was administered for tyrosine kinase activation. Hypoxia (95% N(2) to 5% CO(2)) caused a vasoconstriction either under resting tone or in U46619-precontracted pulmonary veins. Genistein and tyrphostin inhibited hypoxia-induced vasoconstriction both under resting tone and in precontracted veins, while sodium orthovanadate increased these hypoxic contractions. Our findings suggest that tyrosine kinase pathway is involved in hypoxic pulmonary vasoconstriction in sheep isolated pulmonary vein rings.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Genistein; Hypoxia; In Vitro Techniques; Muscle Contraction; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Pulmonary Veins; Sheep; Time Factors; Tyrphostins; Vanadates; Vasoconstriction; Vasoconstrictor Agents

Vasoactive agents were examined in arteries from control rats and rats exposed to intermittent hypoxia (10% oxygen; 8 h/day) for 3, 5 or 20 days. Hypoxic rats developed right ventricular hypertrophy after 5 days, but became pulmonary hypertensive (elevated right ventricular systolic pressure; RVSP) only after 20 days. In pulmonary arteries (main and intralobar), responses to acetylcholine and ionomycin (endothelium-dependent vasodilators) were reduced after 20 and 5 days of intermittent hypoxia, whereas contractions to 5-hydroxytryptamine (5-HT) were enhanced (potency increase >10-fold) after 20, 5 and 3 days. Contractions to endothelin-1 and a thromboxane-mimetic, but not Ca(2+), were also increased. No changes in vascular function occurred in aorta. Since changes in pulmonary vascular function preceded the increase in RVSP they do not result from, but may contribute to, the development of hypoxia-induced pulmonary hypertension. If similar changes occur in humans, they may be important in conditions characterised by intermittent, as opposed to continuous, hypoxia.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetylcholine; Animals; Endothelin-1; Endothelium, Vascular; Hypoxia; In Vitro Techniques; Ionomycin; Male; Muscle, Smooth, Vascular; Potassium Chloride; Pulmonary Artery; Rats; Rats, Wistar; Serotonin; Vasoconstrictor Agents; Vasodilator Agents

Many hemoglobin-based oxygen carriers (HBOCs) produce systemic and pulmonary hypertension and may increase microvascular permeability as a consequence of nitric oxide (NO) scavenging. In this study, we examined the effects of two recombinant human hemoglobin solutions, rHb1.1 and rHb2.0 for injection (rHb2.0), with different rates of NO scavenging on vasoconstrictor reactivity and vascular permeability in isolated, saline-perfused rat lungs. We hypothesized that rHb1.1, a first-generation HBOC with an NO scavenging rate similar to that of native human hemoglobin, would exacerbate pulmonary vasoconstriction and permeability and that rHb2.0, a second-generation HBOC with an NO scavenging rate approximately 20- to 30-fold lower than that of rHb1.1, would minimally influence these responses. Consistent with this hypothesis, rHb1.1 enhanced pulmonary vasoconstrictor reactivity to both hypoxia and thromboxane mimetic U-46619 in a dose-dependent fashion. In contrast, rHb2.0 produced little or no change in reactivity to these stimuli. Furthermore, whereas rHb1.1 abrogated pulmonary vasodilation to the NO-donor S-nitroso-N-acetyl-penicillamine (SNAP), dose-dependent responses to SNAP were preserved, albeit attenuated, in lungs treated with rHb2.0. Finally, the capillary filtration coefficient was unaltered by either rHb1.1 or rHb2.0. We conclude that pulmonary hemodynamic responses to rHb2.0 are greatly reduced compared with those observed with rHb1.1, consistent with rHb2.0 having a diminished capacity to scavenge NO. In addition, neither hemoglobin solution measurably altered microvascular permeability in this preparation.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Capillary Permeability; Dose-Response Relationship, Drug; Free Radical Scavengers; Hemoglobins; Hypoxia; In Vitro Techniques; Lung; Male; Nitric Oxide; Nitric Oxide Donors; Organ Size; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Recombinant Proteins; S-Nitroso-N-Acetylpenicillamine; Solutions; Vascular Resistance; Vasoconstriction; Vasomotor System

Chronic hypoxia alters contractile sensitivity of isolated arteries to alpha-adrenergic stimulation and other agonists. However, most studies have been performed in thoracic aortas or other large vessels making little contribution to vascular resistance in their respective circulations. To determine the effect of chronic hypoxia on the vasoconstrictor response in a small, resistance-sized vessel, we studied second and third generation middle cerebral arteries (MCA; approximately 75-microm internal diameter before mounting). MCA were isolated from normoxic (inspired oxygen = 125 Torr) and hypoxic (8 wk at 3,960 m; inspired oxygen = 90 Torr) guinea pigs, and their vasoconstrictor responses were determined to the thromboxane mimetic U-46619 by using dual-pipette video microscopy. Arteries from hypoxic animals had greater contractile sensitivity to U-46619 compared with those of the normoxic animals (-log EC50 = 7.86 +/- 0.11 vs. 7.62 +/- 0.06, respectively, P < 0.05). Addition of the nitric oxide (NO) inhibitor nitro-L-arginine (200 microM) to the vessel bath eliminated the differences in contractile sensitivity between the MCA from the normoxic and chronically hypoxic groups. Supplementation with L-arginine in the drinking water sufficient to raise plasma L-arginine levels 41% reduced MCA contractile sensitivity to U-46619 in the normoxic group (-log EC50 = 7.22 +/- 0.31, P < 0.05 compared with the nonsupplemented normoxic group) but not in the chronically hypoxic group. These results show that chronic hypoxia increases the sensitivity of the MCA to the vasoconstrictor U-46619, likely because of a reduction in NO production and/or activity.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arginine; Atmosphere Exposure Chambers; Chronic Disease; Dietary Supplements; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guinea Pigs; Hypoxia; In Vitro Techniques; Middle Cerebral Artery; Nitric Oxide; Vasoconstriction; Vasoconstrictor Agents

Female rats develop less severe pulmonary hypertension (PH) in response to chronic hypoxia compared with males, thus implicating a potential role for ovarian hormones in mediating this gender difference. Considering that estrogen upregulates endothelial nitric oxide (NO) synthase (eNOS) in systemic vascular tissue, we hypothesized that estrogen inhibits hypoxic PH by increasing eNOS expression and activity. To test this hypothesis, we examined responses to the endothelium-derived NO-dependent dilator ionomycin and the NO donors S-nitroso-N-acetylpenicillamine and spermine NONOate in U-46619-constricted, isolated, saline-perfused lungs from the following groups: 1) normoxic rats with intact ovaries, 2) chronic hypoxic (CH) rats with intact ovaries, 3) CH ovariectomized rats given 17 beta-estradiol (E(2)beta), and 4) CH ovariectomized rats given vehicle. Additional experiments assessed pulmonary eNOS levels in each group by Western blotting. Our findings indicate that E(2)beta attenuated chronic hypoxia-induced right ventricular hypertrophy, pulmonary arterial remodeling, and polycythemia. Furthermore, although CH augmented vasodilatory responsiveness to ionomycin and increased pulmonary eNOS expression, these responses were not potentiated by E(2)beta. Finally, responses to S-nitroso-N-acetylpenicillamine and spermine NONOate were similarly attenuated in all CH groups compared with normoxic control groups. We conclude that the inhibitory influence of E(2)beta on chronic hypoxia-induced PH is not associated with increased eNOS expression or activity.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Chronic Disease; Endothelium, Vascular; Enzyme Inhibitors; Estradiol; Female; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Ionomycin; Ionophores; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroarginine; Nitrogen Oxides; Ovariectomy; Penicillamine; Polycythemia; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Spermine; Vascular Resistance; Vasoconstrictor Agents; Vasodilation

In pulmonary hypertension, changes in pulmonary vascular structure and function contribute to the elevation in pulmonary artery pressure. The time-courses for changes in function, unlike structure, are not well characterised. Medial hypertrophy and neomuscularisation and reactivity to vasoactive agents were examined in parallel in main and intralobar pulmonary arteries and salt-perfused lungs from rats exposed to hypoxia (10% O2) for 1 and 4 weeks (early and established pulmonary hypertension, respectively). After 1 week of hypoxia, in isolated main and intralobar arteries, contractions to 5-hydroxytryptamine and U46619 (thromboxane-mimetic) were increased whereas contractions to angiotensins I and II and relaxations to acetylcholine were reduced. These alterations varied quantitatively between main and intralobar arteries and, in many instances, regressed between 1 and 4 weeks. The alterations in reactivity did not necessarily link chronologically with alterations in structure. In perfused lungs, constrictor responses to acute alveolar hypoxia were unchanged after 1 week but were increased after 4 weeks, in conjunction with the neomuscularisation of distal alveolar arteries. The data suggest that in hypoxic pulmonary hypertension, the contribution of altered pulmonary vascular reactivity to the increase in pulmonary artery pressure may be particularly important in the early stages of the disease.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetylcholine; Angiotensin I; Angiotensin II; Animals; Hemodynamics; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Lung; Organ Size; Pulmonary Artery; Pulmonary Circulation; Rats; Rats, Wistar; Renin-Angiotensin System; Serotonin; Vasodilator Agents

We tested the hypothesis that the cellular mechanisms mediating hypoxic vasoconstriction (HVC) in frog skin, an important vertebrate respiratory organ, are similar to those mediating HVC in the pulmonary vasculature of mammals. An accepted hypothesis in the lung is that alveolar hypoxia alters the redox potential in vascular smooth muscle cells of arterial vessels. This decreases membrane K+ conductance, causing depolarization. Depolarization increases the open probability of L-type Ca2+ channels, facilitating Ca2+ entry into the cell, which leads to vascular smooth muscle contraction and vasoconstriction. We studied the cutaneous microcirculation of the frog (Xenopus laevis) web by enclosing the web in a transparent chamber that was ventilated with different gas mixtures. Arteriolar and venular diameters were measured by video microscopy. Drugs were applied topically or intravascularly. A dose-dependent constriction to hypoxia occurred in arterioles but not venules, although both vessel types constricted to similar degrees to the thromboxane mimetic U-46619. The magnitude of HVC was not associated with arteriolar size. Constriction of arterioles with 4-amino pyridine, a K+-channel antagonist, was blocked by the L-type Ca2+-channel blocker nifedipine. Nifedipine also antagonized HVC and hypercapnic vasoconstriction. Bay K 8664, a drug that increases the open probability of L-type Ca2+ channels, augmented HVC. These data support our hypothesis that the cellular mechanisms mediating HVC are similar in frog skin and mammalian lungs. This similarity between amphibian and mammalian tissues suggests that the mechanisms of HVC may have arisen relatively early in vertebrate evolution. In addition, because of its structural simplicity and easy accessibility, frog skin may be a useful tissue for studying this general phenomenon in vivo.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; 4-Aminopyridine; Animals; Arterioles; Calcium Channels, L-Type; Homeostasis; Hypoxia; Mammals; Muscle, Smooth, Vascular; Nifedipine; Nitrogen; Oxygen; Potassium Channel Blockers; Skin; Time Factors; Vasoconstriction; Vasoconstrictor Agents; Venules; Xenopus laevis

We tested whether mitochondria function as the O(2) sensor underlying hypoxic pulmonary vasoconstriction (HPV). In buffer-perfused rat lungs, rotenone, myxothiazol, and diphenyleneiodonium, which inhibit mitochondria in the proximal region of the electron transport chain (ETC), abolished HPV without attenuating the response to U46619. Cyanide and antimycin A inhibit electron transfer in the distal region of the ETC, but they did not abolish HPV. Cultured pulmonary artery (PA) myocytes contract in response to hypoxia or to U46619. The hypoxic response was abolished while the response to U46619 was maintained in mutant (rho(0)) PA myocytes lacking a mitochondrial ETC. To test whether reactive oxygen species (ROS) derived from mitochondria act as signaling agents in HPV, the antioxidants pyrrolidinedithiocarbamate and ebselen and the Cu,Zn superoxide dismutase inhibitor diethyldithiocarbamate were used. These abolished HPV without affecting contraction to U46619, suggesting that ROS act as second messengers. In cultured PA myocytes, oxidation of intracellular 2',7'-dichlorofluorescin diacetate (DCFH) dye increased under 2% O(2), indicating that myocytes increase their generation of H(2)O(2) during hypoxia. This was attenuated by myxothiazol, implicating mitochondria as the source of increased ROS during HPV. These results indicate that mitochondrial ATP is not required for HPV, that mitochondria function as O(2) sensors during hypoxia, and that ROS generated in the proximal region of the ETC act as second messengers in the response.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Antimycin A; Antioxidants; Cells, Cultured; Electron Transport; Enzyme Inhibitors; Hypoxia; In Vitro Techniques; Ion Channels; Lung; Methacrylates; Mitochondria; Models, Biological; Muscle, Smooth, Vascular; Onium Compounds; Oxygen; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Rotenone; Signal Transduction; Thiazoles; Uncoupling Agents; Vasoconstriction; Vasoconstrictor Agents

Lung vessel muscularization during hypoxic pulmonary hypertension is associated with local renin-angiotensin system activation. The expression of angiotensin II (Ang II) AT1 and AT2 receptors in this setting is not well known and has never been investigated during normoxia recovery. We determined both chronic hypoxia and normoxia recovery patterns of AT1 and AT2 expression and distal muscularization in the same lungs using in situ binding, reverse transcriptase/polymerase chain reaction, and histology. We also used an isolated perfused lung system to evaluate the vasotonic effects of AT1 and AT2 during chronic exposure to hypoxia with and without subsequent normoxia recovery. Hypoxia produced right ventricular hypertrophy of about 100% after 3 wk, which reversed with normoxia recovery. Hypoxia for 2 wk was associated with simultaneous increases (P<0.05) in AT1 and AT2 binding (16-fold and 18-fold, respectively) and in muscularized vessels in alveolar ducts (2. 8-fold) and walls (3.7-fold). An increase in AT2 messenger RNA (mRNA) (P<0.05) was also observed, whereas AT1 mRNA remained unchanged. After 3 wk of hypoxia, muscularization was at its peak, whereas all receptors and transcripts showed decreases (P<0.05 versus hypoxia 2 wk for AT1 mRNA), which became significant after 1 wk of normoxia recovery (P<0.05 versus hypoxia 2 wk). Significant reversal of muscularization (P<0.01) was found only after 3 wk of normoxia recovery in alveolar wall vessels. Finally, the AT1 antagonist losartan completely inhibited the vasopressor effect of Ang II in hypoxic and normoxia-restored lungs, whereas the AT2 agonist CGP42112A had no effect. Our data indicate that in lungs, chronic hypoxia-induced distal muscularization is associated with early and transient increases in AT2 and AT1 receptors probably owing to hypoxia- dependent transcriptional and post-transcriptional regulatory mechanisms, respectively. They also indicate that the vasotonic response to Ang II is mainly due to the AT1 subtype.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Antihypertensive Agents; Gene Expression Regulation, Developmental; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Imidazoles; Iodine Radioisotopes; Ligands; Losartan; Male; Muscle, Smooth, Vascular; Oligopeptides; Oxygen; Pulmonary Alveoli; Pulmonary Artery; Pyridines; Radioligand Assay; Rats; Rats, Wistar; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Receptors, Angiotensin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Vasoconstrictor Agents

Hypoxic pulmonary vasoconstriction (HPV) is essential for matching lung perfusion with ventilation, thus optimizing pulmonary gas exchange. Preceding studies provided evidence for a role of both nitric oxide (NO) and superoxide/ H(2)O(2) formation in this vasoregulatory mechanism. Both agents might be operative via stimulation of guanylate cyclase with formation of the vasodilatory cyclic guanosine monophosphate (cGMP), the loss of which under conditions of hypoxia contributes to HPV. This view is challenged by the recent suggestion of increased rather than decreased superoxide/H(2)O(2) formation in hypoxia. We addressed the role of NO-dependent versus NO-independent guanylate cyclase activity in hypoxic and pharmacologically evoked vasoconstriction in perfused rabbit lungs. Two inhibitors of soluble guanylate cyclase, LY83583 (2 to 16 microM) and methylene blue (20 to 60 microM), increased baseline pulmonary artery pressure under normoxic conditions and markedly amplified the vasoconstrictor response to both hypoxia and the stable thromboxane analogue U46619. Under conditions of preblocked lung NO synthesis (N(G)-mono-methyl-L-arginine), however, additional guanylate cyclase inhibition further enhanced the vasoconstrictor response to U46619 but did not influence the strength of HPV. The selective phosphodiesterase V inhibitor Zaprinast (1 to 10 microM), used for prolongation of the cGMP half-life, reduced the hypoxia-induced pressor response to a larger extent than the pressor response to U46619. This difference was lost under conditions of preblocked NO synthesis. Equilibration of the lung perfusate with molecular NO suppressed the HPV more potently than the U46619-induced vasoconstrictor response. We conclude that NO-dependent guanylate cyclase activity has an important role in attenuating the vasoconstrictor response to alveolar hypoxia in rabbit lungs. In contrast, no evidence was obtained for a role of NO-independent cGMP formation in HPV. In this feature, HPV differs from that elicited by the thromboxane analogue U46619.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Aminoquinolines; Animals; Aspirin; Blood Pressure; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Female; Guanylate Cyclase; Hypoxia; In Vitro Techniques; Lung; Male; Methylene Blue; Nitric Oxide; omega-N-Methylarginine; Pulmonary Artery; Purinones; Rabbits; Vasoconstriction; Vasoconstrictor Agents

Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion with ventilation. Controversy exists whether decreased or increased reactive oxygen species may elicit HPV and from which source such oxygen metabolites are derived. In rabbit lungs, we detected transcripts of a nonphagocytic NADPH oxidase subunit homologous to mitogenic oxidase-1 (Mox1) or NADPH oxidase homolog 1 (NOH-1L). In perfused rabbit lungs, we employed 1) a new NADPH oxidase inhibitor [4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF; 100-600 microM)] and 2) the superoxide dismutase (SOD) inhibitors diethyldithiocarbamic acid (DETC; 100 microM to 10 mM) and triethylenetetramine (TETA; 1-25 mM). Specificity of these agents for HPV was investigated by comparison with U-46619-induced vasoconstrictions. AEBSF induced a transient increase in pulmonary arterial pressure with increased strength of HPV. Subsequent to this initial response, normoxic pulmonary arterial pressure was not affected and HPV was specifically suppressed. Whereas DETC turned out to act in a nonspecific fashion, TETA suppressed HPV specifically. These findings provide evidence of a role for a nonphagocytic NAD(P)H oxidase with superoxide and SOD-related hydrogen peroxide formation in HPV. Because HPV was inhibited but not mimicked by the inhibitors, increased rather than decreased superoxide and/or hydrogen peroxide formation is suggested as the hypoxia-provoked signaling event.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Blood Pressure; Ditiocarb; Female; Hydrogen Peroxide; Hypoxia; In Vitro Techniques; Lung; Male; NADPH Oxidases; Pulmonary Circulation; Rabbits; Reactive Oxygen Species; Sulfones; Transcription, Genetic; Trientine; Vasoconstriction

Chronic hypoxic pulmonary hypertension (PH) is associated with vasoconstriction and structural remodeling of pulmonary vessels including narrowing of the arterial lumen and loss of distal functional arteries. To test whether lung overexpression of the angiogenic factor vascular endothelial growth factor (VEGF) is beneficial in hypoxic PH, recombinant adenovirus encoding the human VEGF 165 gene under the control of a cytomegalovirus promoter (Ad. VEGF) or control vector containing no gene in the expression cassette (Ad.Null) was administered intratracheally to rats. With Ad. VEGF (10(8) plaque-forming units [pfu]), VEGF protein was present in bronchoalveolar lavage fluid as early as 2 d and until 17 d after gene transfer, but was not detected in serum. Only small patchy areas of mononuclear cells without cell damage, edema, or hemorrhage were observed on lung histology with no significant change in lung permeability. In rats pretreated with Ad.VEGF (10(8) pfu) 2 d before a 2-wk exposure to hypoxia (10% O(2)), lower values versus Ad. Null-pretreated controls were found for pulmonary artery pressure (25 +/- 1 versus 30 +/- 2 mm Hg, P < 0.05), right ventricular over left ventricular-plus-septum weight (0.37 +/- 0.01 versus 0.47 +/- 0. 02, P < 0.001), normalized wall thickness of 50- to 200-microm vessels (P < 0.001), and muscularization of distal vessels (P < 0. 001). Pretreatment with Ad.VEGF (10(8) pfu) increased endothelial nitric oxide synthase activity in lung tissue and partially restored endothelium-dependent vasodilation in isolated lungs from chronically hypoxic rats, as assessed by improvement of ionophore A23187-induced vasodilation and attenuation of endothelin-1 (300 pmol)-induced vasoconstriction, an effect abolished in the presence of nitro-L-arginine methylester. We conclude that adenoviral-mediated VEGF overexpression in the lungs attenuates development of hypoxic PH, in part by protecting endothelium-dependent function.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenoviridae; Animals; Bronchoalveolar Lavage Fluid; Calcimycin; Capillary Permeability; DNA, Recombinant; Dose-Response Relationship, Drug; Endothelial Growth Factors; Endothelin-1; Gene Expression Regulation; Gene Transfer, Horizontal; Humans; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Ionophores; Lung; Lymphokines; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Rats; Rats, Wistar; Time Factors; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Vasodilation

Previous studies suggest that inducible (i) nitric oxide synthase (NOS) expression within the pulmonary vasculature is increased in rats with chronic hypoxia (CH)-induced pulmonary hypertension. We therefore hypothesized that enhanced iNOS expression associated with CH causes attenuated pulmonary vasoconstrictor responsiveness. To test this hypothesis, we examined the effect of selective iNOS blockade with L-N6-(1-iminoethyl)lysine dihydrochloride (L-NIL) and nonselective NOS inhibition with Nomega-nitro-L-arginine (L-NNA) on vasoconstrictor responses to U-46619 in isolated saline-perfused lungs from both control and CH (4 wk at 380 mmHg) rats. We additionally measured pulmonary hemodynamic responses to L-NIL in conscious CH rats (fraction of inspired O2 = 0.12). Finally, iNOS mRNA levels were assessed in lungs from each group of rats using ribonuclease protection assays. Despite a significant increase in iNOS mRNA expression after exposure to CH, responses to U-46619 were unaltered by L-NIL but augmented by L-NNA in lungs from both control and CH rats. Pulmonary hemodynamics were similarly unaltered by L-NIL in conscious CH rats. We conclude that iNOS does not modulate pulmonary vasoconstrictor responsiveness after long-term hypoxic exposure.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Chronic Disease; Enzyme Inhibitors; Hemodynamics; Hypoxia; In Vitro Techniques; Lipopolysaccharides; Lung; Lysine; Male; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Vascular Resistance; Vasoconstriction; Vasoconstrictor Agents

Alkalosis-induced relaxation was measured in precontracted arterial rings from 1-wk-old piglets exposed to normoxia or to 3 days of chronic hypoxia. In normoxic piglet arteries, alkalosis-induced relaxation was blunted in arteries without functional endothelium and in arteries treated with nitric oxide synthase or guanylate cyclase inhibitors but not in arteries treated with cyclooxygenase inhibitors or Ca2+- and ATP-dependent K+-channel inhibitors. Inhibition of voltage-dependent K+ channels with 10(-3) M 4-aminopyridine also failed to block alkalosis-induced relaxation. 4-Aminopyridine at 10(-2) M did block the response, but this may have been due to sustained vascular smooth muscle depolarization. Arteries from hypoxic piglets exhibited greater contraction to the thromboxane mimetic U-46619, decreased endothelium-dependent relaxation, and blunted alkalosis-induced relaxation. The residual relaxation was eliminated by nitric oxide synthase but not by cyclooxygenase or voltage-dependent K+-channel inhibition. Alkalosis-induced relaxation of newborn piglet pulmonary arteries appears to be mediated by the nitric oxide-cGMP pathway and is attenuated after 3 days of hypoxia, likely because of decreased nitric oxide activity.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Alkalosis; Animals; Animals, Newborn; Chronic Disease; Endothelium, Vascular; Enzyme Inhibitors; Guanylate Cyclase; Hypoxia; Nitric Oxide Synthase; Potassium Channel Blockers; Pulmonary Artery; Reference Values; Swine; Vasoconstrictor Agents; Vasodilation

We previously demonstrated augmented endothelium-derived nitric oxide (EDNO)-dependent pulmonary arterial dilation and increased arterial endothelial nitric oxide synthase (eNOS) levels in chronic hypoxic (CH) and monocrotaline (nonhypoxic) models of pulmonary arterial hypertension. Therefore, we hypothesized that the long-term elevation of arterial eNOS levels associated with CH is related to pulmonary hypertension or some factor(s) associated with hypertension and not directly to hypoxia. To test this hypothesis, we examined responses to the EDNO-dependent dilator ionomycin in U-46619-constricted, isolated, saline-perfused lungs from control rats, CH (4 wk at 380 mmHg) rats, and rats previously exposed to CH but returned to normoxia for 4 days or 2 wk. Microvascular pressure was assessed by double-occlusion technique, allowing calculation of segmental resistances. In addition, vascular eNOS immunoreactivity was assessed by quantitative immunohistochemistry, and eNOS mRNA abundance was determined by RT-PCR assays. Our findings indicate that 4-day and 2-wk posthypoxic rats exhibit persistent pulmonary hypertension, likely due to maintained arterial remodeling and polycythemia associated with prior exposure to CH. Furthermore, arterial dilation to ionomycin was augmented in lungs from each experimental group compared with controls. Finally, arterial eNOS immunoreactivity and whole lung eNOS mRNA levels remained elevated in posthypoxic animals. These findings suggest that altered vascular mechanical forces or vascular remodeling contributes to enhanced EDNO-dependent arterial dilation and upregulation of arterial eNOS in various models of established pulmonary hypertension.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Chronic Disease; Gases; Gene Expression Regulation; Hematocrit; Hemodynamics; Hypertrophy, Right Ventricular; Hypoxia; In Vitro Techniques; Ionomycin; Lung; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Vascular Resistance

1. Hypoxia (PO2 < 5 mmHg) decreased vessel tone in isolated rat mesenteric arteries precontracted with either high [K+] or the thromboxane analogue U46619. This response was not altered by N-nitro-L-arginine (L-NA) and indomethacin. 2. Simultaneous measurement of pHi and tension showed that the decrease in vessel tone was accompanied by an intracellular acidification. Similar reductions in tone and pHi were observed with the metabolic inhibitors 2,4-dinitrophenol (DNP) and sodium azide. 3. The presence of the lactate transport inhibitor alpha-cyano-4-hydroxy-cinnamic acid (CHC) increased the magnitude of the acidification and resulted in a significantly faster reduction in tone in response to hypoxia. Addition of CHC to normoxic tissues caused both a vasodilatation and a reduction of pHi. 4. A decrease in pHi induced on washout of ammonium chloride (NH4Cl) resulted in an increase in tone. 5. Relaxation to hypoxia or metabolic inhibition was unaffected when the change in pHi was neutralized by addition of the weak base trimethylamine (TMA). 6. It is concluded that severe hypoxia decreases tone in isolated rat mesenteric arteries by a mechanism which is independent of nitric oxide and prostaglandins. Both severe hypoxia and metabolic inhibition reduced pHi, although this does not appear to be contributing to the changes in tone observed.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 2,4-Dinitrophenol; Ammonium Chloride; Animals; Antimetabolites; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Male; Mesenteric Arteries; Methylamines; Muscle Contraction; Muscle Relaxation; Nitric Oxide; Potassium; Rats; Rats, Wistar; Uncoupling Agents; Vasodilation

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

After an initial vasodilator response to alkalosis, many children with pulmonary hypertension exhibit marked pulmonary vascular reactivity despite continued alkalosis therapy. This study sought to a) identify the mediator of alkalosis-induced pulmonary vasodilation in isolated lamb lungs; b) determine whether alkalosis-induced pulmonary vasodilation decreases over time in this model; and c) determine whether alkalosis enhanced vascular reactivity to subsequent pressor stimuli.. Prospective, interventional study.. Isolated perfused lungs from 1-month-old lambs.. Hypocarbic alkalosis, hypoxia, and infusion of the thromboxane mimetic agent U46619. Pulmonary artery pressure was measured at constant flow, so a change in pressure reflects change in resistance. Hypoxic pulmonary artery pressure was compared after 20 and 100 mins of hypocarbic alkalosis or normocarbia in control and cyclooxygenase-inhibited lungs. Pulmonary artery dose responses to U46619 were then measured in control lungs. Responses to hypoxia and U46619 were also compared after 60-80 mins of hypocarbic or normocarbic normoxia. Hypocarbic alkalosis acutely reduced hypoxic pulmonary vascular resistance, and this was sustained for at least 100 mins. Cyclooxygenase inhibition blocked this vasodilation, suggesting that it was mediated by dilator prostaglandins. However, subsequent reactivity to U46619 was enhanced in hypoxic alkalotic lungs, and both hypoxia and U46619 caused significant vasoconstriction in normoxic alkalotic lungs.. Alkalosis caused sustained vasodilation when pulmonary vascular resistance was high but either failed to attenuate or enhanced vascular reactivity to subsequent pressor stimuli.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Alkalosis, Respiratory; Animals; Cyclooxygenase Inhibitors; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Indomethacin; Prospective Studies; Pulmonary Circulation; Random Allocation; Sheep; Time Factors; Vascular Resistance; Vasoconstrictor Agents; Vasodilation

The reactivity of pulmonary veins during adaptation from pre- to postnatal life is not well characterized. With an in vitro organ bath technique, the responses to the contractile and relaxant agonists U-46619 (10(-10) to 3 x 10(-6) M) and acetylcholine (10(-9) to 10(-4) M) were compared in adjacent conduit pulmonary vein and artery rings from 66 piglets aged 1 wk preterm to 14 days of postnatal life and from adult tissue. Five additional piglets were made hypertensive by exposure to chronic hypoxia for 3 days after birth. Both arteries and veins showed smaller contractile and relaxant responses before birth than after. By 5 min after birth, the contraction by arteries and relaxation by veins had increased (P < 0.05). By 3 days of age, arterial relaxation increased, but in all animals, venous relaxation exceeded that in arteries (P < 0.05). Veins contracted more than arteries in animals aged 3-14 days. Neonatal hypoxia diminished the responses to both agonists in the veins (P < 0.05), whereas the response in the arteries remained similar to that in the normal newborn. We speculate that veins may be more important in postnatal adaptation than previously suggested.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetylcholine; Adaptation, Physiological; Age Factors; Animals; Animals, Newborn; Hypertension, Pulmonary; Hypoxia; Oxygen; Pulmonary Artery; Pulmonary Veins; Swine; Vasoconstriction; Vasoconstrictor Agents; Vasodilator Agents

The anorexiant dexfenfluramine, which inhibits 5-hydroxytryptamine (5-HT) uptake, has been associated with an increase in the relative risk of developing primary pulmonary hypertension. The aim of this study was to investigate in rats whether dexfenfluramine (1) alters the pulmonary vasomotor effects of 5-HT and (2) aggravates the development of pulmonary hypertension during exposure to various levels of chronic hypoxia. In isolated lungs from normoxic rats, dexfenfluramine up to 10(-4) M did not elicit any vasoactive effects, and neither did pretreatment with dexfenfluramine (10[-5] M in the perfusate) modify the vasoactive effects of 5-HT. In normoxic conscious rats, dexfenfluramine given intravenously potentiated the pulmonary pressor response to acute hypoxia (10% O2). In rats chronically treated with dexfenfluramine during a 2-wk exposure to 15% or 10% O2, plasma 5-HT concentrations were significantly increased compared with hypoxic controls, whereas no differences were found for pulmonary artery pressure, right ventricular hypertrophy, or pulmonary vessel muscularization. In contrast, a continuous 5-HT infusion providing a sustained increase in plasma 5-HT levels was associated with increased muscularization of distal pulmonary arteries in response to 10% O2. Simultaneous administration of dexfenfluramine prevented the effect of exogenous 5-HT on vascular remodeling. Our findings show that dexfenfluramine does not potentiate the development of pulmonary hypertension in rats exposed to chronic hypoxia, despite its effect on plasma 5-HT concentrations.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Appetite Depressants; Blood Pressure; Fenfluramine; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Male; Pulmonary Circulation; Rats; Rats, Wistar; Selective Serotonin Reuptake Inhibitors; Serotonin; Vasoconstriction; Vasoconstrictor Agents

Down-regulation of the endogenous nitric oxide (NO) pathway may explain rebound pulmonary hypertension after discontinuation of inhaled NO. We determined whether the prolonged administration of inhaled NO increases pulmonary vasoconstriction, which may occur from decreased endogenous NO. Rats were placed in normoxic (N; 21% O2) or hypoxic (H; 10% O2) chambers with or without inhaled NO (20 ppm) for 1 or 3 wk. Immediately after or 24 h after discontinuation of NO, vasoconstrictive responses were determined in isolated lungs to acute hypoxia (HPV; 0% O2 for 6 min), angiotensin II (0.05 microg), and the thromboxane analog U-46619 in the presence and absence of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; 100 microM). Inhaled NO did not alter HPV or angiotensin II vasoconstriction in the N group immediately after or 24 h after discontinuation of NO. In the H group, inhaled NO decreased HPV but had no effect on the angiotensin II vasoconstriction compared with H alone. Inhaled NO did not alter the response to L-NAME. Inhaled NO did not alter, whereas L-NAME significantly decreased, the dose of U-46619 required to increase the pulmonary pressure by 10 mm Hg. In conclusion, prolonged inhaled NO decreased or did not alter HPV and did not alter vasoconstriction secondary to angiotensin II, U-46619, or L-NAME in N and H rats. These results suggest that prolonged inhaled NO does not increase pulmonary vasoconstriction, as would be expected from down-regulation of endogenous NO.. High pulmonary pressure has been observed clinically after discontinuation of inhaled NO. This rat study suggests that 1-3 wk of inhaled NO does not increase pulmonary vasoconstriction, as would be expected from decreasing the endogenous vasodilator NO.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Administration, Inhalation; Angiotensin II; Animals; Hypoxia; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Time Factors; Vasoconstriction; Vasoconstrictor Agents; Vasodilator Agents

Inhaled nitric oxide (iNO) is being used to treat pulmonary hypertension in a variety of chronic lung diseases associated with pulmonary vascular remodeling. We hypothesized that chronic hypoxia (CH)-induced vascular remodeling decreases the vasodilatory effectiveness of iNO due to a thickened diffusional barrier. We therefore examined segmental vasodilatory responses to iNO in U-46619-constricted lungs isolated from control and CH (4 weeks at 0.5 atm) rats using double occlusion technique. We further measured lung fluid flux and vascular wall thickness in lungs from each group to provide an index of vascular permeability and vascular remodeling, respectively. CH was associated with decreased venous, but not arterial, responsiveness to iNO in saline-perfused lungs. In addition, the presence of red blood cells (RBC) within the perfusate greatly reduced venodilation in both groups of lungs, indicating that venous responsiveness to iNO in saline-perfused lungs is largely dependent upon transport of NO from an upstream site. In contrast, RBC had no effect on arterial dilation in control lungs, but attenuated arterial dilation to iNO in lungs from CH rats. Finally, fluid flux and arterial wall thickness were greater in lungs from CH rats. We conclude that arterial remodeling associated with CH may limit venous dilation to iNO. Furthermore, the decreased arterial responsiveness to iNO following CH is consistent with extravasation of hemoglobin within the arterial vasculature.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Administration, Inhalation; Animals; Blood Vessels; Body Fluids; Chronic Disease; Erythrocytes; Hypoxia; In Vitro Techniques; Lung; Male; Nitric Oxide; Perfusion; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Vascular Resistance; Vasoconstrictor Agents; Vasodilator Agents

The aim of this study was to investigate the potential role of 5-hydroxytryptamine (5-HT) on development of pulmonary hypertension during chronic exposure to mild (15% O2) and severe (10% O2) hypoxia. In isolated lungs from normoxic rats preconstricted with U-46619, 5-HT (10(-12)-10(-8) M) induced dose-dependent vasodilation (n = 6), which was suppressed by the NO synthesis inhibitor nitro-L-arginine methyl ester (L-NAME, 10(-4) M, n = 5) and reduced by the 5-HT3-receptor antagonist MDL-7222 (10(-5) M, n = 6). The vasoconstriction that was observed with higher concentrations of 5-HT (10(-7)-10(-4) M) was inhibited by ketanserin (10(-5) M) and methiothepin (10(-5) M, n = 6 each). The vasodilator response to 5-HT was suppressed in lungs from rats exposed to 10% O2 but not 15% O2 (n = 6 each). In conscious rats, intravenous administration of 5-HT potentiated the pulmonary pressor response to acute hypoxia (10% O2, n = 5), an effect that remained unchanged after pretreatment with a 5-HT1 and a 5-HT2 antagonist (n = 4) but was attenuated after treatment with the cyclooxygenase inhibitor meclofenamate (n = 4). Treatment with 5-HT (5 nmol/h i.v. by osmotic pumps) for 2 wk in rats simultaneously exposed to 10% O2 increased pulmonary arterial pressure, right ventricular hypertrophy, and muscularization of pulmonary vessels in comparison with their hypoxic controls (n = 12 each). No changes occurred in 15% O2 hypoxic rats (n = 12 each). The present findings show that 5-HT potentiates development of pulmonary hypertension in rats exposed to chronic hypoxia.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Cyclooxygenase Inhibitors; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Infusions, Intravenous; Lung; Male; Meclofenamic Acid; NG-Nitroarginine Methyl Ester; Prostaglandin Endoperoxides, Synthetic; Pulmonary Circulation; Rats; Rats, Wistar; Receptors, Serotonin; Receptors, Serotonin, 5-HT3; Serotonin; Serotonin Antagonists; Thromboxane A2; Tropanes; Vasoconstriction; Vasodilation

We have previously demonstrated that arterial, but not venous, vasodilatory responses to endothelium-derived nitric oxide (EDNO)-dependent agonists are enhanced in lungs isolated from rats with chronic hypoxia (CH)-induced pulmonary arterial hypertension. These data suggest that CH is associated with increased endothelial nitric oxide synthase (eNOS) activity within the pulmonary arterial vasculature. In addition, the correlation of increased pulmonary arterial pressure with selectively enhanced arterial responsiveness to EDNO-mediated agonists suggests that arterial hypertension, rather than hypoxia per se, is a contributing factor in this response. Therefore, we hypothesized that 1) CH selectively upregulates eNOS within the pulmonary arterial vasculature and 2) monocrotaline (MC)-induced pulmonary arterial hypertension selectively enhances pulmonary arterial dilation to EDNO-dependent dilators and upregulates arterial eNOS. We examined the responses to the EDNO-dependent dilators arginine vasopressin and ionomycin in U-46619-constricted isolated perfused lungs from control and MC-treated rats. Microvascular pressure was assessed by the double-occlusion technique, allowing calculation of segmental resistances. Lungs from MC-treated rats exhibited augmented arterial dilation to arginine vasopressin compared with control lungs. However, the responses to ionomycin were not different between the two groups. Quantitative immunocytochemistry was used to compare pulmonary eNOS immunoreactivity in vessels from control, CH, and MC-treated rats. eNOS staining was more intense in the arteries of CH and MC-treated rats compared with those of control animals, whereas CH and MC treatment had no effect on eNOS staining in veins. We conclude that pulmonary arterial hypertension, or altered vascular mechanical forces associated with hypertension, may be responsible for the augmented EDNO-dependent arterial dilation and upregulation of arterial eNOS in lungs from CH and MC-treated rats.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arginine Vasopressin; Blood Pressure; Chronic Disease; Endothelium, Vascular; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Ionomycin; Lung; Male; Monocrotaline; Nitric Oxide Synthase; Prostaglandin Endoperoxides, Synthetic; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Thromboxane A2; Vascular Resistance; Vasoconstrictor Agents

We have recently reported in normal isolated-perfused rat lungs that low basal tone appears to be regulated by nitric oxide (NO)-dependent and -independent mechanisms of soluble guanylate cyclase activation. In this study, we examined the role of NO in the regulation of pulmonary artery (PA) tone from rats with renin-dependent hypertension. Rats were made hypertensive by ligating the abdominal aorta above the left and below the right renal artery (aortic coarctation, AC). Mean arterial pressure significantly increased from 119 +/- 8.4 mmHg in control animals to 156 +/- 15 mmHg 7-14 days after AC surgery. PA pressures, however, remained unchanged (8.5 +/- 3.4 mmHg in control animals vs. 11 +/- 3.3 mmHg in AC animals). Hypoxic contractions in U-46619 precontracted isolated small PA (160-260 microns diameter) were significantly increased from 51 +/- 13 mg in the control group to 142 +/- 38 mg (P < or = 0.05) in AC animals. Nitro-L-arginine (NLA; 100 microM) contractions were also enhanced in the AC animal. The enhanced NLA response may correlate with an increase in endothelial cell NO synthase (NOS) as detected by Western blotting (132 +/- 28% of control; P < 0.05). These data suggest that, in this renin-dependent model of systemic hypertension, there is increased endothelial cell NOS activity that maintains low PA tone, preventing the lung from developing increased pressures.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aorta, Abdominal; Aortic Coarctation; Blood Pressure; Endothelium, Vascular; Guanylate Cyclase; Hypertension; Hypoxia; In Vitro Techniques; Male; Muscle Contraction; Muscle Tonus; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitroarginine; Prostaglandin Endoperoxides, Synthetic; Pulmonary Artery; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Regression Analysis; Renin; Thromboxane A2; Vasoconstrictor Agents

We have previously demonstrated that chronic hypoxia (CH) augments pulmonary arterial dilation to the endothelium-derived nitric oxide (EDNO)-dependent pulmonary vasodilator arginine vasopressin (AVP). The present study examined 1) whether this enhanced vasoreactivity is observed with other agents that act by stimulating constitutive NO synthase (cNOS), 2) whether CH increases arterial vascular smooth muscle sensitivity to NO, and 3) whether endogenous endothelin (ET) or an endothelium-derived hyperpolarizing factor (EDHF) contributes to this altered arterial reactivity following CH. We examined responses to the receptor-mediated EDNO-dependent dilators histamine and ET-1, the nonreceptor-mediated EDNO-dependent dilator ionomycin, and the NO donors 1, 3-propanediamine, N-4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino] butyl (spermine NONOate) and S-nitroso-N-acetylpenicillamine (SNAP) in U-46619-constricted, isolated perfused lungs from control and CH rats. Additional experiments examined responses to AVP in the presence of the ET-receptor antagonist PD-145065 or the K+ channel blockers glibenclamide or tetraethylammonium (TEA) in lungs from each group. Microvascular pressure was assessed by double occlusion, allowing calculation of segmental resistances. Total and arterial vasodilatory responses to histamine, ET-1, and ionomycin were augmented in lungs from CH vs. control animals. However, CH did not alter the vasodilation to spermine NONOate or SNAP. PD-145065, glibenclamide, and TEA had no effect on responses to AVP in either group. We conclude that increased activity of arterial cNOS may be responsible for the augmented pulmonary arterial dilation to EDNO-dependent vasodilators following CH.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Biological Factors; Endothelins; Endothelium, Vascular; Histamine; Hypoxia; Ionomycin; Ionophores; Male; Muscle, Smooth, Vascular; Nitric Oxide; Oligopeptides; Prostaglandin Endoperoxides, Synthetic; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Thromboxane A2; Vasoconstrictor Agents; Vasodilation

Others have shown that inhaled nitric oxide causes reversal of pulmonary hypertension in anaesthetized perinatal sheep. The present study examined haemodynamic responses to inhaled NO in the normal and constricted pulmonary circulation of unanaesthetized newborn lambs. Three experiments were conducted on each of 7 lambs. First, to determine a minimum concentration of NO which could reverse acute pulmonary hypertension caused by infusion of the thromboxame mimic U46619, the haemodynamic effects of 5 different doses of inhaled NO were examined. Second, the effects of inhaling 80 ppm NO during hypoxic pulmonary vasoconstriction were examined. Finally, to determine if tachyphalaxis occurs during NO inhalation, lambs were exposed to 80 ppm NO for 3 h during which time pulmonary arterial pressure was doubled by infusion of U46619. Breathing NO (80 ppm) caused a slight but significant decrease in pulmonary vascular resistance (PVR) in lambs with normal pulmonary arterial pressure (PAP). Nitric oxide, inhaled at concentrations between 10 and 80 ppm for 6 min (F1O2 = 0.60), caused decreases in PVR when PAP was elevated with U46619. Nitric oxide acted selectively on the pulmonary circulation, i.e. no changes occurred in systemic arterial pressure or any other measured variable. Breathing 80 ppm NO for 6 min reversed hypoxic pulmonary vasoconstriction. In the chronic exposure study, inhaling 80 ppm NO for 3 h completely reversed U46619-induced pulmonary hypertension. Although arterial methaemoglobin increased during the 3-h exposure to 80 ppm NO, there was no indication that this concentration of NO impairs oxygen loading. These data demonstrate that NO, at concentrations as low as 10 ppm, is a potent, rapid-action, and selective pulmonary vasodilator in unanaesthetized newborn lambs with elevated pulmonary tone. Furthermore, these data support the use of inhaled NO for treatment of infants with pulmonary hypertension.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Administration, Inhalation; Animals; Animals, Newborn; Dose-Response Relationship, Drug; Drug Administration Schedule; Hypertension; Hypertension, Pulmonary; Hypoxia; Nitric Oxide; Prostaglandin Endoperoxides, Synthetic; Sheep; Thromboxane A2; Vasoconstrictor Agents; Vasodilator Agents

The effect of in vitro hypoxia for 1 h on concentration-response curves to vasoconstrictor spasmogens was examined in rat isolated pulmonary arteries. Hypoxia, like levcromakalim (KATP channel opener), did not affect contractions to endothelin-1 but attenuated contractions to U46619 ((1,5,5,)-hydroxy-11 alpha, 9 alpha-epoxymethano) prosta 5Z, 13E-dienoic acid; thromboxane-mimetic), angiotensin II, noradrenaline and 5-hydroxytryptamine. The attenuation was prevented by glybenclamide. In pre-contracted arteries, subsequent exposure to hypoxia caused a response consisting of four phases (transient relaxation due to endothelium-derived nitric oxide; transient contraction; slow relaxation; sustained contraction). Glybenclamide, if added before hypoxia, did not eliminate either of the relaxant phases but, if added during the sustained contractile phase, caused further contraction. We conclude that exposure of pulmonary arteries to prolonged hypoxia causes KATP channels to open, as in systemic arteries; this diminishes contractions to some, but not all, vasoconstrictor spasmogens. The data suggest that endothelin-1, unlike other vasoconstrictors, would remain a highly effective pulmonary vasoconstrictor under severe hypoxic conditions.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Benzopyrans; Cromakalim; Drug Interactions; Glyburide; Hypoxia; In Vitro Techniques; Male; Potassium Channel Blockers; Potassium Channels; Prostaglandin Endoperoxides, Synthetic; Pulmonary Artery; Pyrroles; Rats; Rats, Wistar; Thromboxane A2; Vasoconstrictor Agents

In this study, we investigated the hemodynamic effects and receptor-blocking properties of the nonselective endothelin antagonist bosentan in pigs during normoxia and acute hypoxia. Hypoxic pulmonary hypertension was induced by decreasing the fraction of inhaled oxygen to 0.1. In a control group of pigs, hemodynamic parameters proved to be stable through 2 hours of hypoxia. Infusions of endothelin-1, endothelin-3, and sarafotoxin 6c into the pulmonary artery resulted in pulmonary and systemic vasoconstriction during normoxia, whereas endothelin administration during hypoxic pulmonary hypertension resulted in pulmonary vasodilation. After administration of bosentan, the vasopressor effect of endothelin-1 during normoxia was significantly attenuated and the pulmonary vasodilatory effect of endothelin-1 during hypoxia was reduced. Furthermore, the development of hypoxic pulmonary hypertension was significantly reduced by bosentan. In contrast, bosentan did not influence the pulmonary vasopressor response to the thromboxane mimic U-46619. We therefore conclude that vasopressor endothelin receptors seem to be activated by endogenous endothelin released during hypoxia, leading to an increase in the pulmonary vascular tone.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Bosentan; Cardiac Output; Endothelin Receptor Antagonists; Endothelins; Hemodynamics; Hypertension, Pulmonary; Hypoxia; Oxygen; Prostaglandin Endoperoxides, Synthetic; Pulmonary Artery; Sulfonamides; Swine; Thromboxane A2; Vascular Resistance; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Viper Venoms

The present study was designed to point out similarities between the effects on pulmonary circulation caused by hypoxia and by a chemoreceptor stimulant (S1867, an almitrine analog). Isolated rat lungs were perfused at a constant flow with homologous blood and ventilated under normoxic, hypoxic or hyperoxic conditions. (1) At 0.25 microgram/ml, S1867 potentiated the hypoxic pressor response, while at 1 microgram/ml, it induced a significant increase in pulmonary artery pressure (PAP) at 21% O2. (2) Since the expression of an oxygen-binding protein (NADPH-oxidase like) has been demonstrated in the rat carotid bodies, we studied the effects of the NADPH-oxidase inhibitor diphenyleneiodonium (DPI) on HPV and on S1867-induced pulmonary vascular responses. Both responses were totally abolished by DPI (40 microM), whereas the vasoconstriction induced by a thromboxane A2 analog (U46619) remained unchanged. (3) Vascular responses to hypoxia and S1867 (1 microgram/ml) were both reversed by a bolus of the sulfhydryl oxidant diamide (3 mg). (4) The S1867-induced response was prevented and reversed by the supply of inhaled oxygen, which was without action on the vasoconstriction induced by U46619. These results suggest that the almitrine analog and hypoxia act at least partly through the same cellular mechanism, involving a DPI-sensitive protein.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Almitrine; Animals; Blood Pressure; Chemoreceptor Cells; Disease Models, Animal; Enzyme Inhibitors; Hypoxia; In Vitro Techniques; Male; NADPH Oxidases; Nitric Oxide Synthase; Onium Compounds; Perfusion; Prostaglandin Endoperoxides, Synthetic; Pulmonary Artery; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Respiratory System Agents; Thromboxane A2; Vasoconstriction

1. Angiotensin II (AII) binding density and the effect of chronic AII receptor blockade were examined in the rat model of hypoxia-induced pulmonary hypertension. 2. [125I]-[Sar1,Ile2]AII binding capacity was increased in lung membranes from rats exposed to hypoxia (10% fractional inspired O2) for 7 days compared to normal rats (Bmax 108 +/- 12 vs 77 +/- 3 fmol mg-1 protein; P < 0.05), with no significant change in dissociation constant. Competition with specific AII receptor subtype antagonists demonstrated that AT1 is the predominant subtype in both normal and hypoxic lung. 3. Rats treated intravenously with the AT1 antagonist, GR138950C, 1 mg kg-1 day-1 rather than saline alone during 7 days of exposure to hypoxia developed less pulmonary hypertension (pulmonary arterial pressure: 21.3 +/- 1.7 vs 28.3 +/- 1.1 mmHg; P < 0.05), right ventricular hypertrophy (right/left ventricle weight ratio: 0.35 +/- 0.01 vs 0.45 +/- 0.01; P < 0.05) and pulmonary artery remodelling (abundance of thick-walled pulmonary vessels: 9.6 +/- 1.4% vs 20.1 +/- 0.9%; P < 0.05). 4. The reduction in cardiac hypertrophy and pulmonary remodelling with the AT1 antagonist was greater than that achieved by a dose of sodium nitroprusside (SNP) that produced a comparable attenuation of the rise in pulmonary arterial pressure during hypoxia. 5. The data suggest that AII, via the AT1 receptor, has a role in the early pathogenesis of hypoxia-induced pulmonary hypertension in the rat.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Angiotensin II; Angiotensin Receptor Antagonists; Animals; Benzofurans; Hypertension, Pulmonary; Hypoxia; Lung; Male; Nitroprusside; Prostaglandin Endoperoxides, Synthetic; Rats; Rats, Wistar; Receptors, Angiotensin; Thromboxane A2

We designed experiments to determine in vitro the influence of hypoxia on endothelium function in porcine pulmonary artery and vein. Rings of large isolated intralobar pulmonary arteries and veins were mounted in organ chambers for isometric tension recording. In veins and arteries with endothelium (precontracted with histamine and U 46619, respectively), hypoxia induced a transient contractile response that was significantly greater in veins. These contractile responses were fully blocked by L-nitro arginine (LNA) in arteries but only partially in veins. In pulmonary vessels without endothelium, only venous rings produced a contractile response to hypoxia; this contraction was blocked by indomethacin. In precontracted pulmonary arterial and venous rings, bradykinin (BK) induced endothelium-dependent relaxations unaffected by indomethacin. Venous relaxations were fully blocked by LNA, but arterial relaxations were only partially inhibited by the nitric oxide (NO) synthase inhibitor. In KCl (30 mM)-precontracted vessels, the endothelium-dependent relaxations were minimally affected in veins but significantly inhibited in arteries. Identical results were obtained in tissue contracted with histamine or U 46619 in presence of tetraethyl ammonium (TEA 10 mM). Hypoxia (30 mm Hg) abolished the venous relaxation but did not significantly influence the arterial relaxation. In arterial rings, the effects of KCl and LNA (or hypoxia) were additive. These results suggest that in isolated porcine pulmonary veins, endothelium-dependent relaxation to BK is exclusively dependent on NO formation. In arteries, however, NO production is partially involved. Another mechanism, possibly endothelium-dependent hyperpolarization, exists. These differences in endothelial responsiveness lead to different patterns of response to hypoxia.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arginine; Bradykinin; Endothelium, Vascular; Female; Hypoxia; In Vitro Techniques; Indomethacin; Male; Nitric Oxide; Nitroarginine; Potassium Channels; Prostaglandin Endoperoxides, Synthetic; Pulmonary Artery; Pulmonary Veins; Swine; Tetraethylammonium Compounds; Thromboxane A2; Vasoconstrictor Agents; Vasodilation

Nitric oxide generation and hypoxic vasoconstriction in buffer-perfused rabbit lungs. J. Appl. Physiol. 78(4): 1509-1515, 1995.--We investigated the role of nitric oxide (NO) generation in hypoxic pulmonary vasoconstriction in buffer-perfused rabbit lungs. Exhaled NO was detected by chemiluminescence, and intravascular NO release was quantified as perfusate accumulation of nitrite, peroxynitrite, and nitrate (NOx). Under baseline conditions, exhaled NO was 45.3 +/- 4.1 parts per billion (1.8 +/- 0.2 nmol/min), and lung NOx release into the perfusate was 4.1 +/- 0.4 nmol/min. Alveolar hypoxia (alveolar PO2 of approximately 23 Torr) induced readily reproducible pressor responses preceded by a sharp drop in exhaled NO concentration. In contrast, perfusate NOx accumulation was not affected. Vasoconstrictor responses to U-46619 and angiotensin II were not accompanied by a decrease in NO exhalation. NG-monomethyl-L-arginine dose-dependently suppressed NO exhalation and amplified pressor responses to hypoxia > U-46619 and angiotensin II. In conclusion, portions of baseline NO generation originating from sites with ready access to the gaseous space sharply decrease in response to alveolar hypoxia, whereas the intravascular release of NO is unchanged. Such differential regulation of lung NO synthesis in response to hypoxia may suggest a complex role in the regulation or modulation of hypoxic pulmonary vasoconstriction.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Angiotensin II; Animals; Arginine; Female; Hypoxia; In Vitro Techniques; Lung; Male; Nitric Oxide; omega-N-Methylarginine; Perfusion; Prostaglandin Endoperoxides, Synthetic; Pulmonary Circulation; Rabbits; Thromboxane A2; Vasoconstriction

Both increases and decreases in endothelium-derived nitric oxide (EDNO) activity have been described in the developing pulmonary vasculature. We hypothesized that differences in baseline vasomotor tone and/or oxygen tension may contribute to this variability. Pulmonary arterial dose responses to endothelium-dependent and -independent vasodilators acetylcholine (ACh) and sodium nitroprusside (SNP), respectively, were measured in indomethacin-treated lungs of 1- to 2-day-old (2D) and 1-mo-old (1M) lambs. During 4% O2 ventilation, baseline pulmonary vascular resistance (PVR) and the dilator response to both ACh and SNP were greater in 2D lungs. However, when baseline PVR values were matched at both ages during either hypoxia or infusion of a thromboxane mimetic under normoxic conditions, developmental differences in ACh-induced vasodilation were minimal. Furthermore, hypoxia itself did not alter the responses to ACh in 2D lungs. In contrast, SNP caused greater vasodilation in 2D than in 1M lungs regardless of baseline PVR. These data and studies suggest that whereas high PVR enhances EDNO synthesis, responsiveness to ENDO decreases as synthesis of ENDO increases in developing lungs studied under basal conditions.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetylcholine; Age Factors; Animals; Animals, Newborn; Blood Gas Analysis; Endothelium, Vascular; Hypoxia; Infusions, Intravenous; Nitric Oxide; Nitroprusside; Perfusion; Prostaglandin Endoperoxides, Synthetic; Pulmonary Artery; Pulmonary Gas Exchange; Sheep; Thromboxane A2; Vascular Resistance; Vasoconstrictor Agents; Vasodilator Agents

HA1004, an isoquinolinesulfonamide and a cyclic nucleotide-dependent protein kinase inhibitor, is an intracellular calcium antagonist that produces vascular smooth muscle (VSM) relaxation in vitro. We studied the hemodynamic effects of intravenous (i.v.) infusions of HA1004 (0.1-2.0 mg/kg) in vivo in 8 newborn lambs, at rest and during pulmonary hypertension induced either by the i.v. infusion of U46619, a thromboxane A2 (TXA2) mimic, or by alveolar hypoxia. For comparison, we also studied the hemodynamic effects of i.v. infusions of nifedipine (15 and 40 micrograms/kg/min), a calcium entry blocker. At rest, HA1004 produced slight but significant changes in pulmonary and systemic arterial pressure (PAP, SAP) and pulmonary and systemic vascular resistances (PVR, SVR) (p < 0.05). During pulmonary hypertension induced by U46619, HA1004 decreased PAP 12-23% and PVR 9-33% (p < 0.05), whereas SAP decreased 7% and SVR decreased 14% at only one dose (p < 0.05). During pulmonary hypertension induced by alveolar hypoxia, HA1004 decreased PAP 6-32% and PVR 11-30% (p < 0.05), whereas SAP decreased 15% only at the highest dose (p < 0.05). Linear regression analysis of the pooled data demonstrated that HA1004 caused selective pulmonary vasodilation during pulmonary hypertension. Nifedipine decreased PAP 6 and 14% and SAP 5 and 17% during pulmonary hypertension. In newborn lambs with pulmonary hypertension, HA1004, an intracellular calcium antagonist, is more selective and potent than nifedipine, a calcium entry blocker, in decreasing PAP and therefore may be useful in treatment of children with pulmonary hypertension.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Animals, Newborn; Calcium Channel Blockers; Hemodynamics; Hypertension, Pulmonary; Hypoxia; Infusions, Intravenous; Isoquinolines; Muscle Relaxation; Nifedipine; Prostaglandin Endoperoxides, Synthetic; Protein Kinase Inhibitors; Pulmonary Alveoli; Regression Analysis; Sheep; Sulfonamides; Thromboxane A2

Nitric oxide (NO) is a potent endogenous vasodilator. Its role in the normal and stressed pulmonary circulation is unclear. To better understand the importance of endogenous NO in normal physiological responses, we studied the effects of altered NO availability on the change in pulmonary vascular tone that accompanies exercise. In paired studies we measured blood flow and pressures in the pulmonary circulation at rest and during treadmill exercise at a speed of 4 mph with and without (a) N omega-nitro-L-arginine, 20 mg/kg intravenously, a selective inhibitor of NO synthase; (b) L-arginine, 200 mg/kg intravenously, substrate for NO synthase; (c) combination of the inhibitor and substrate; and (d) inhalation of NO > 30 ppm, to determine if endogenous release of NO elicits maximal vasodilation. In addition, we sought to determine the site of NO effect in the pulmonary circulation by preconstriction with either U-44619 or hypoxia (fraction of inspired O2 = 0.12) using a distal wedged pulmonary catheter technique. NO synthase inhibition raised pulmonary vascular tone equally at rest and exercise. L-Arginine reversed the effects of NO synthase inhibition but had no independent effect. NO inhalation did not reduce pulmonary vascular tone at rest or enhance the usual reduction in pulmonary vascular resistance with exercise. The effect of NO synthase inhibition was in pulmonary vessels upstream from small veins, suggesting that endogenous NO dilates primarily small arteries and veins at rest. We conclude that, in sheep, endogenous NO has a basal vasodilator function that persists during, but is not enhanced by, exercise.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Amino Acid Oxidoreductases; Animals; Arginine; Blood Pressure; Hemodynamics; Hypoxia; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Physical Conditioning, Animal; Prostaglandin Endoperoxides, Synthetic; Pulmonary Circulation; Sheep; Thromboxane A2; Vascular Resistance; Vasoconstriction; Vasoconstrictor Agents; Vasodilation

Chronic hypoxic exposure elicits pulmonary vascular remodeling and may alter normal pulmonary endothelial function. We examined the vasodilatory response to the receptor-mediated endothelium-dependent dilator arginine vasopressin (AVP), the non-receptor-mediated endothelium-dependent dilator A-23187, and the nitric oxide (NO) donor sodium nitroprusside in lungs isolated from control or chronically hypoxic rats. Lungs were isolated from male Sprague-Dawley rats and perfused with a physiological saline solution containing 4% albumin. Arterial and venous pressures were monitored and microvascular pressure was estimated by double occlusion, allowing assessment of segmental resistances. After equilibration, lungs were constricted with the thromboxane mimetic U-46619. Upon development of a stable pressor response, lungs were dilated with one of the above agents. A series of doses of AVP was administered to separate groups of lungs from control or chronically hypoxic rats. Lungs from chronically hypoxic rats exhibited an augmented dilatory response to AVP compared with control lungs, and this effect was due to enhanced dilation of precapillary segments. The total and segmental vasodilatory responses to A-23187 and sodium nitroprusside were not different between the two groups of lungs, suggesting that chronic hypoxia did not upregulate the enzyme NO synthase or enhance the vascular smooth muscle responsiveness to NO. Thus our data suggest that the augmented total and pulmonary arterial dilation to AVP after chronic hypoxia is most likely due to altered receptor-mediated processes of the hormone.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Amino Acid Oxidoreductases; Animals; Arginine Vasopressin; Calcimycin; Capillary Resistance; Dose-Response Relationship, Drug; Hypoxia; Male; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Perfusion; Prostaglandin Endoperoxides, Synthetic; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Receptors, Vasopressin; Thromboxane A2; Vasodilation

The possible involvement of ATP-sensitive K+ channels (KATP) in hypoxic relaxation of isolated porcine coronary arteries was investigated. Tubular segments taken from the left anterior descending artery were suspended in myographs for recording of isometric contractile force. Hypoxia (pO2 = 20.3 mm Hg +/- 0.5) produced a greater relaxation in preparations contracted by 30 mM K+ (49.7% +/- 7.2) compared with 124 mM K+ (19.9% +/- 2.2) which is compatible with the involvement of K+ channel activation in the mechanism of hypoxic relaxation. In a normal glucose-containing Krebs solution the KATP blocker glibenclamide (1 microM) failed to influence the hypoxic relaxation of preparations contracted by the thromboxane A2 analogue U-46619. Under conditions created to inhibit non-oxidative ATP production from glycolysis using a glucose-free Krebs solution containing 2-deoxyglucose (10 mM), the hypoxic relaxation was enhanced from 54.5% +/- 5.0 to 77.2% +/- 4.4. Under these conditions glibenclamide (1 microM) significantly inhibited the hypoxic relaxant response from 77.2% +/- 4.2 to 55.2% +/- 4.4 and prolonged the time until half-maximal relaxation from 5.5 min +/- 0.6 to 8.1 min +/- 0.6. A low concentration of the KATP opener levcromakalim (30 nM) failed to significantly potentiate the hypoxic relaxation. The adenosine receptor blocker theophylline (1 microM) or removal of the endothelium showed no effect on the hypoxic relaxation. In normal glucose-containing Krebs solution, indomethacin (10 microM) caused a small but significant inhibition of the hypoxic relaxation from 54.5% +/- 5.0 to 41.6% +/- 3.6.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Triphosphate; Animals; Benzopyrans; Coronary Vessels; Cromakalim; Deoxyglucose; Endothelium, Vascular; Glyburide; Glycolysis; Hypoxia; In Vitro Techniques; Indomethacin; Isometric Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Prostaglandin Endoperoxides, Synthetic; Pyrroles; Swine; Theophylline; Thromboxane A2; Vasoconstrictor Agents; Vasodilator Agents

The thromboxane mimetic U46619 (11 alpha,9 alpha-epoxymethano PGH2) increased 45Ca2+ uptake in sheep coronary artery rings. A larger increase occurred in endothelium-denuded than in endothelium-inact rings (increase in 45Ca2+ uptake: endothelium intact = 9.6 +/- 3.8, endothelium-denuded = 33.2 +/- 8.1 nmol g-1), in agreement with the increase in U46619 contraction produced by endothelium denudation. Hypoxia (PO2 = 4 mm Hg) inhibited both the U46619 contraction (at the U46619 EC50, contraction under oxygenated conditions = 158 +/- 35, under hypoxic conditions = 105 +/- 40 g cm-2) and the U46619-stimulated 45Ca2+ uptake. It is concluded that inhibition, by hypoxia, of the U46619 contractile effect on sheep coronary artery rings may be in part due to inhibition of U46619-stimulated 45Ca2+ uptake.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Calcium; Calcium Radioisotopes; Coronary Vessels; Endothelium, Vascular; Hypoxia; In Vitro Techniques; Isometric Contraction; Muscle, Smooth, Vascular; Oxygen Consumption; Prostaglandin Endoperoxides, Synthetic; Sheep; Thromboxane A2; Vasodilator Agents

Coronary artery contractility is well known to be modulated by oxygen partial pressure. Both smooth muscle and the endothelium contribute to coronary artery oxygen sensitivity. Mechanisms underlying endothelium-dependent effects of oxygen include the sensitivity of the nitric oxide/endothelium-derived relaxing factor (EDRF), hydrogen peroxide, and eicosanoid pathways. In the present study, we characterize a novel endothelium-dependent component of porcine coronary artery oxygen sensitivity that is independent of these known pathways. Porcine coronary arteries were stimulated with either KCl or U46619. Hypoxia elicited a transient increase in force that was much greater in endothelium-intact arteries. This effect was abolished by nitric oxide/EDRF pathway inhibitors NG-monomethyl-L-arginine and N-nitro-L-arginine. In the steady state, hypoxia reduced isometric force to a similar degree in both intact and denuded arteries. Reoxygenation elicited a rapid and transient relaxation only in intact arteries. In contrast, this endothelium-dependent relaxation was not inhibited by nitric oxide/EDRF pathway inhibitors nor inhibitors of other potential oxygen-sensitive pathways, such as indomethacin, aminotriazole, superoxide dismutase, catalase, propranolol, or ouabain. The reoxygenation relaxation was, however, sensitive to very low levels of oxygen and was inhibited by cyanide and rotenone, suggesting an involvement of mitochondrial metabolism. Interestingly, the relaxation response to reoxygenation, similar to that for substance P, could be restored in denuded arteries by coupling with an endothelium-intact donor artery. This "sandwich" experiment suggests that the endothelium dependence is mediated by a transmissible factor. Our results indicate that a novel class of endothelium-dependent factors may contribute to coronary artery responses to changes in oxygen partial pressure.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arginine; Coronary Vessels; Endothelium, Vascular; Hypoxia; Oxygen; Partial Pressure; Potassium Chloride; Prostaglandin Endoperoxides, Synthetic; Swine; Thromboxane A2; Vasoconstrictor Agents; Vasodilation

The vascular endothelium mediates, in part, pulmonary vascular tone. Because endothelin-1 (ET-1), a paracrine hormone produced by vascular endothelial cells, has vasoactive properties, we investigated the hemodynamic effects of intrapulmonary injections of ET-1 in eight intact newborn lambs at rest and during pulmonary hypertension. At rest, ET-1 (50-1,000 ng/kg) did not change pulmonary arterial pressure. During pulmonary hypertension induced by the infusion of U46619 (a thromboxane A2 mimic), ET-1 (50-1,000 ng/kg) produced a selective dose-dependent decrease in pulmonary arterial pressure (5.8 +/- 3.9 to 32.9 +/- 6.9%; P < 0.05). Similarly, during pulmonary hypertension induced by alveolar hypoxia, ET-1 (50-500 ng/kg) produced a selective dose-dependent decrease in pulmonary arterial pressure (7.2 +/- 3.6 to 26.1 +/- 3.3%; P < 0.05). The decrease in pulmonary arterial pressure produced by ET-1 (250 ng/kg) was attenuated by N omega-nitro-L-arginine (an inhibitor of endothelium-derived nitric oxide synthesis, 23.7 +/- 3.4 vs. 12.5 +/- 4.7%; P < 0.05) and by glibenclamide (an ATP-gated potassium-channel blocker, 25.2 +/- 5.0 vs. 9.6 +/- 5.3%; P < 0.05) but not by meclofenamic acid (an inhibitor of prostaglandin synthesis). ET-1 is a pulmonary vasodilator during pulmonary hypertension in the intact newborn lamb. The vasodilating properties are mediated, in part, by release of endothelium-derived nitric oxide, and by activation of ATP-gated potassium channels.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Animals, Newborn; Arginine; Endothelins; Glyburide; Hypertension, Pulmonary; Hypoxia; Injections, Intra-Arterial; Meclofenamic Acid; Nitroarginine; Prostaglandin Endoperoxides, Synthetic; Pulmonary Artery; Pulmonary Circulation; Rest; Sheep; Vasodilation

To investigate dilator effects of endothelins (ETs) on the pulmonary circulation and possible changes induced by chronic hypoxia, we examined vascular responses to ET-1 and ET-3 as well as ET binding to receptor subtypes ETA and ETB in the lungs from rats exposed to either room air (controls), hypoxia (10% O2) for 3 wk (3 WH), or 3 WH followed by recovery to room air (3 WH+R). In controls, both ETA and ETB receptor binding was present in smooth muscle of airways and vessels. Infusion of ET-1 or ET-3 (3-100 pM) to isolated perfused lungs preconstricted by U-46619 produced dose-dependent vasodilation with a greater potency of ET-3 (P < 0.01). The vasodilator responses to ET-1 and ET-3 were potentiated by the cyclooxygenase blocker meclofenamate (3 x 10(-6) M) or by the thromboxane synthetase inhibitor R-68070. In meclofenamate-treated lungs, the vasodilator responses to ET-1 and ET-3 remained unaffected by the inhibitor of nitric oxide synthesis, NG-monomethyl-L-arginine (5 x 10(-4) M) or by the guanylate cyclase inhibitor, methylene blue (10(-4) M). Conversely, the K+ channel blockers glibenclamide (10(-4) M) and tetraethylammonium (10(-4) M) attenuated the vasodilator responses to both ET-1 and ET-3. The selective ETA receptor antagonist BQ-123 did not alter ET-induced vasodilation, whereas it attenuated ET-induced vasoconstriction. Vasodilation to both ET-1 and ET-3 was abolished in lungs from 3 WH rats (P < 0.01) but was fully restored in lungs from 3 WH+R rats. Pulmonary vasodilation induced by the K+ channel opener pinacidil, which was suppressed by glibenclamide, did not differ between controls and 3 WH rat lungs. We found no change in ETA and ETB receptor binding from pulmonary vessels in H rat lungs compared with controls. In conclusion, endothelin-induced pulmonary vasodilation which may involve activation of K+ channels is abolished during chronic hypoxia. This abolition does not appear to be related to alterations in ET-receptor subtypes or to unresponsiveness of K+ channels in the pulmonary circulation.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arginine; Autoradiography; Dose-Response Relationship, Drug; Endothelins; Glyburide; Guanidines; Hypoxia; In Vitro Techniques; Iodine Radioisotopes; Lung; Male; Meclofenamic Acid; Nitroarginine; Pentanoic Acids; Pinacidil; Potassium Channel Blockers; Prostaglandin Endoperoxides, Synthetic; Pulmonary Circulation; Pyridines; Rats; Rats, Wistar; Receptors, Endothelin; Tetraethylammonium; Tetraethylammonium Compounds; Thromboxane A2; Thromboxane-A Synthase; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

There is increasing evidence that resting pulmonary vascular tone is mediated by the release of endothelium-derived relaxing factors (EDRF). However, the importance of EDRF release during pulmonary hypertension is unknown. Therefore, in eight newborn lambs we studied the effects of both N omega-nitro-L-arginine (an inhibitor of EDRF synthesis) and L-arginine (a precursor of EDRF synthesis) during pulmonary hypertension induced either by the intravenous infusion of U-46619 (a thromboxane A2 mimic) or by hypoxia. After pretreatment with N omega-nitro-L-arginine, the increases in pulmonary arterial pressure produced by U-46619 (102.0 +/- 34.9% vs. 144.8 +/- 28.6%, P less than 0.05) and by hypoxia (35.6 +/- 17.3% vs. 91.4 +/- 24.8%, P less than 0.05) were significantly augmented. However, after pretreatment with L-arginine, the increases in pulmonary arterial pressure produced by U-46619 (107.0 +/- 21.4% vs. 62.6 +/- 22.6%, P less than 0.05) and hypoxia (44.3 +/- 18.3% vs. 9.2 +/- 11.7%, P less than 0.05) were significantly attenuated. These results suggest that during pulmonary hypertension, EDRF is released to limit the increase in pulmonary arterial pressure and that L-arginine availability becomes rate limiting for further EDRF synthesis and release.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Animals, Newborn; Arginine; Blood Pressure; Cardiac Output; Heart Rate; Hypertension, Pulmonary; Hypoxia; Nitric Oxide; Nitroarginine; Prostaglandin Endoperoxides, Synthetic; Pulmonary Circulation; Sheep; Vascular Resistance; Vasoconstrictor Agents

The influence of methylene blue, an inhibitor of soluble guanylate cyclase, on responses to ventilatory and precapillary hypoxia was investigated in the intact-chest cat under conditions of controlled blood flow and constant left atrial pressure. Because methylene blue increased vascular tone, responses to hypoxia were compared when lobar arterial pressure was raised to similar levels with U 46619 and with methylene blue. When lobar arterial pressure was raised with U 46619, ventilation with 7.5% O2 increased lobar arterial pressure significantly. Infusion of methylene blue in concentrations that raised lobar arterial pressure to a value similar to that attained with U 46619 prevented the pressor response to hypoxia, and a significant depressor response was unmasked. The depressor response to hypoxia in the methylene blue-treated animal was not altered by meclofenamate but was blocked by propranolol. A reduction in lobar arterial perfusate PO2 induces an increase in pulmonary vascular resistance in the cat, and this response was prevented by methylene blue. During methylene blue infusion, the vasodilator response to acetylcholine was reduced, whereas the response to isoproterenol was not altered. Although the response to hypoxia was prevented, the pressor response to prostaglandin F2 alpha was not changed. The response to ventilatory hypoxia was enhanced by propranolol or ICI 118551, suggesting that the response is modulated by circulating catecholamines that are probably of adrenal origin. The effects of methylene blue on vascular tone and responses to hypoxia and acetylcholine were reversible, and responses returned to control value after the infusion was terminated.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetylcholine; Adrenergic beta-Antagonists; Animals; Cats; Dinoprost; Female; Hypoxia; Male; Meclofenamic Acid; Methylene Blue; Perfusion; Propranolol; Prostaglandin Endoperoxides, Synthetic; Pulmonary Circulation; Respiration; Vasoconstriction; Vasodilation

The pulmonary vascular reactivity to endothelin-1 (ET-1) was assessed in rats previously exposed to 11% O2 (hypoxic) or room air (controls) for 3 weeks. In isolated control lung preparations studied during conditions of increased tone by U46619 (50 pmol/min) and treated with meclofenamate (3 microM), low doses of ET-1 (30 and 100 pM) reduced the pressor response to U46619 by 58 +/- 5% (p less than 0.01). Vasodilation induced by ET-1 was not abolished by the antagonist of endothelium-dependent relaxing factor (EDRF) NG-monomethyl-L-arginine (5 x 10(-4) M), which suppressed vasodilator response to ionophore A23187 (10(-8)-10(-7) M). Higher doses of ET-1 (300 and 1,000 pM) induced vasoconstriction during conditions of basal tone, and the pressor response to 300 pM ET-1 was enhanced by EDRF antagonists. Administration of ET-1 to lungs from hypoxic rats failed to cause pulmonary vasodilation and instead induced a greater pulmonary pressor response (300 pM) than in control rat lungs (7 +/- 1.5 vs. 1.6 +/- 0.5 mm Hg, p less than 0.01), which was not further potentiated by EDRF antagonists. Infusion of 300 pM ET to conscious catheterized animals induced a sustained increase in pulmonary resistance only in the hypoxic group (from 305 +/- 37 to 389 +/- 55 mm Hg/L/min, p less than 0.01) (n = 7). The results suggest that depending on the dose, ET-1 can cause pulmonary vasodilation (independent of EDRF release) or vasoconstriction (opposed by EDRF). During chronic hypoxic pulmonary hypertension, ET-1 behaves only as a pulmonary vasoconstrictor.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arginine; Calcimycin; Endothelins; Hemodynamics; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Male; Muscle Tonus; Nitric Oxide; omega-N-Methylarginine; Prostaglandin Endoperoxides, Synthetic; Pulmonary Circulation; Rats; Rats, Inbred Strains; Vasodilation

We investigated the effects of infusions of ATP-MgCl2 on the circulation in 11 spontaneously breathing newborn lambs during pulmonary hypertension induced either by the infusion of U-46619, a thromboxane A2 mimetic, or by hypoxia. During pulmonary hypertension induced by U-46619, ATP-MgCl2 (0.01-1.0 mg.kg-1.min-1) caused a significant dose-dependent decrease in pulmonary arterial pressure (12.4-40.7%, P less than 0.05), while systemic arterial pressure decreased only at the highest doses (P less than 0.05). Left atrial infusions of ATP-MgCl2 caused systemic hypotension without decreasing pulmonary arterial pressure. During hypoxia-induced pulmonary hypertension, ATP-MgCl2 caused a similar significant dose-dependent decrease in pulmonary arterial pressure (12.0-41.1%, P less than 0.05), while systemic arterial pressure decreased only at high doses (P less than 0.05). Regression analysis showed selectivity of the vasodilating effects of ATP-MgCl2 for the pulmonary circulation during pulmonary hypertension induced either by U-46619 or hypoxia. ATP-MgCl2 is a potent vasodilator with a rapid metabolism that allows for selective vasodilation of the vascular bed first encountered (pulmonary or systemic). We conclude that infusions of ATP-MgCl2 may be clinically useful in the treatment of children with pulmonary hypertension.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Triphosphate; Animals; Animals, Newborn; Blood Pressure; Hypertension, Pulmonary; Hypoxia; Prostaglandin Endoperoxides, Synthetic; Pulmonary Circulation; Rest; Sheep; Vasodilation

1. The effects of calcitonin gene-related peptide (CGRP) and neuropeptide Y (NPY) were examined on sheep circumflex (2-2.5 mm o.d.) coronary artery rings, with and without endothelium, under oxygenated, hypoxic and simulated ischaemic conditions. The interaction between the vasoconstrictor effects of NPY and the thromboxane mimetic, U46619, was also studied. 2. Ischaemia was simulated by modification of the composition of the physiological salt solution by increasing potassium and H+, including lactate and reducing glucose and PO2. 3. Hypoxia alone and simulated ischaemia increased the maximum vasodilatation produced by CGRP. CGRP (30 nM) abolished and markedly reduced the contraction that was induced by hypoxia and simulated ischaemia respectively. 4. Hypoxia increased and simulated ischaemia reduced the contractile response to NPY in endothelium intact rings. When the endothelium was removed, NPY caused a contraction under ischaemic conditions only. The hypoxic and ischaemic-induced contractions were augmented by NPY (30 nM). 5. In the rings containing endothelium, NPY enhanced the contraction caused by U46619 during hypoxia only. In endothelium-denuded preparations, NPY increased or partially restored the contraction caused by U46619. 6. These results show that the responsiveness of coronary artery rings isolated from sheep to either CGRP or NPY is modified by hypoxia or simulated myocardial ischaemia.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Calcitonin Gene-Related Peptide; Coronary Disease; Coronary Vessels; Endothelium, Vascular; Hypoxia; In Vitro Techniques; Muscle, Smooth, Vascular; Neuropeptide Y; Potassium Chloride; Prostaglandin Endoperoxides, Synthetic; Sheep

1. The role(s) of the endothelium in modulating the responsiveness of isolated circumflex coronary artery rings (o.d. = 2.0-2.5 mm and o.d. = 0.6-1.3 mm) from sheep was investigated under oxygenated and hypoxic conditions. 2. Removal of the endothelium abolished the contraction produced by lowering the PO2 from 620 to 8 mmHg (either under optimal resting tension or precontracted by 40 mM KCl). In denuded artery rings sudden hypoxia caused relaxation. 3. Under oxygenated conditions, removal of the endothelium augmented the vasoconstrictor effects of U46619, 5-hydroxytryptamine (5-HT) and K+. In the denuded artery rings, hypoxia abolished the contractile effects of U46619 and reduced the contractile effects of 5-HT and K+. 4. Under oxygenated conditions, the vasorelaxant effect of adenosine was depressed by removal of the endothelium. In endothelium-denuded preparations, the small relaxant effect of adenosine remaining was greatly potentiated. 5. Haemolysate (1 microliter ml-1) caused an endothelium-dependent contraction under oxygenated conditions. The hypoxic contraction observed in the artery ring under resting tension was significantly potentiated by haemolysate (1 microliter ml-1). Haemolysate 1 microliter ml-1 had no effect on the denuded artery rings under hypoxic conditions. 6. Haemolysate (1 microliter ml-1) potentiated the vasoconstrictor effects of U46619 (0.5 microM), 5-HT (1 microM) and K+ (24 mM) under oxygenated conditions. 7. These results indicate that endothelium profoundly modifies the effect of hypoxia on the responsiveness of sheep isolated left circumflex coronary artery rings.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine; Animals; Cats; Coronary Vessels; Endothelium, Vascular; Hemolysis; Hypoxia; In Vitro Techniques; Isometric Contraction; Muscle, Smooth, Vascular; Nitroprusside; Potassium; Prostaglandin Endoperoxides, Synthetic; Serotonin; Sheep; Substance P; Vasoconstrictor Agents

Thromboxane A2 (TxA2) is an arachidonic acid metabolite which causes severe pulmonary vasoconstriction (PV) and may mediate the PV produced by platelet-activating factor (PAF-acether) and leukotriene D4 (LTD4). To determine the role of TxA2 receptors on PAF-acether, LTD4, and hypoxia-induced PV, we administered PAF-acether 0.1 nmol/kg, the TxA2 analog U-46619 0.2 micrograms/kg/min, LTD4 3.0 micrograms/kg, or acute hypoxia (FiO2 = 0.12 for 3 min) before and during the infusion of the selective TxA2 receptor blocker SQ 29,548 50 micrograms/kg/min or vehicle into 27 open-chest, anesthetized newborn piglets, measuring pulmonary and systemic arterial pressures, cardiac index, and right and left ventricular pressures and dimensions. Mean pulmonary arterial pressure rose and cardiac index fell in response to PAF-acether (14 +/- 1 to 32 +/- 2 mm Hg and 91 +/- 5 to 15 +/- 5 mL/kg/min, both p less than 0.01), U-46619 (11 +/- 1 to 28 +/- 2 mm Hg and 93 +/- 10 to 36 +/- 9 mL/kg/min, both p less than 0.01), and LTD4 (13 +/- 3 to 22 +/- 2 mm Hg and 85 +/- 12 to 29 +/- 9 mL/kg/min, both p less than 0.05). Acute hypoxia increased PAP (12 +/- 1 to 26 +/- 2 mm Hg, p less than 0.01) but did not alter cardiac index. Infusion of SQ 29,548 prevented PAF-acether and U-46619-induced increases in pulmonary arterial pressure (13 +/- 1 to 14 +/- 1 mm Hg and 12 +/- 1 to 12 +/- 1 mm Hg) and decreases in cardiac index (70 +/- 4 to 70 +/- 3 mL/kg/min and 94 +/- 14 to 92 +/- 12 mL/kg/min) but failed to alter the response to LTD4 or hypoxia. Vehicle had no effect. We conclude that TxA2 receptors are not involved in LTD4 or hypoxia-induced PV but play an important role in the PV produced by PAF-acether and U-46619.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Animals, Newborn; Blood Pressure; Bridged Bicyclo Compounds, Heterocyclic; Fatty Acids, Unsaturated; Female; Hemodynamics; Hydrazines; Hypoxia; Lung; Male; Platelet Activating Factor; Prostaglandin Endoperoxides, Synthetic; Swine; Thromboxane A2; Vasoconstriction

1. The effects of low oxygen tension on tone and on the responsiveness to contractile and relaxant agents were examined on circumflex coronary artery rings isolated from sheep. 2. When artery rings (2-2.5 mm o.d.) were set at their optimal resting tension, introduction of hypoxia (0% O2) caused a sustained contraction which was reversible on washing with oxygenated Krebs solution. In precontracted (40 mM KCl) arteries, hypoxia caused a similar response except that it was preceded by a transient relaxation. 3. The hypoxia-induced contraction was potentiated by the combination of phentolamine (1 microM) and propranolol (1 microM), markedly reduced by verapamil (10 microM) and either abolished or reduced by indomethacin (1 microM). Indomethacin itself caused a contraction. 4. Under hypoxic conditions, the contractile effects of U46619 (a stable thromboxane analogue) and 5-hydroxytryptamine (5-HT) and the vasodilator effects of noradrenaline, iloprost (a prostacyclin mimetic) and adenosine were markedly potentiated. In contrast, vasoconstriction to potassium or acetylcholine was depressed. 5. Changing the gases from 95% O2 to 12% O2 had no significant effect on the contractile effects of U46619. However, the maximum contractile effect of U46619 was significantly enhanced by changing the gases from 12% O2 to 0% O2. 6. Rings from a smaller branch (0.6-1.3 mm o.d.) of the circumflex coronary artery of the sheep, in the presence of hypoxia, exhibited qualitatively similar changes in the responsiveness to U46619, 5-HT and adenosine to those observed in the large artery. However, the effect of potassium was potentiated rather than depressed. 7. It is concluded that hypoxia-induced contraction may involve a modified release of cyclooxygenase products and be partly dependent upon the availability of extracellular calcium. 8. The change in the responsiveness of coronary arteries, under hypoxia, to both constrictor and dilator mediators may have clinical relevance to myocardial ischaemia and angina pectoris.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Coronary Vessels; Hypoxia; In Vitro Techniques; Indomethacin; Muscle Contraction; Muscle, Smooth, Vascular; Oxygen Consumption; Phentolamine; Potassium Chloride; Propranolol; Prostaglandin Endoperoxides, Synthetic; Serotonin; Sheep; Verapamil

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetophenones; Animals; Animals, Newborn; Bridged Bicyclo Compounds, Heterocyclic; Fatty Acids, Unsaturated; Hydrazines; Hypoxia; Lipids; Platelet Activating Factor; Prostaglandin Endoperoxides, Synthetic; Pulmonary Circulation; SRS-A; Swine; Tetrazoles; Thromboxane A2; Vasoconstriction

Thromboxane A2 has been implicated as a mediator of cardiorespiratory dysfunction in sepsis. This study evaluated whether or not thromboxane A2 was necessary or sufficient for these adverse effects to occur during bacteremia. Fourteen adult swine under barbiturate anesthesia and breathing room air were monitored with arterial and pulmonary artery catheters. Animals were studied for 4 hours in three groups: group I, graded infusion of 10(9)/ml Aeromonas hydrophila; group II, Aeromonas hydrophila infusion plus SQ 29,548 (thromboxane A2 antagonist); and group III, U46619 (thromboxane A2 agonist) infusion in normal swine to pulmonary artery pressures observed in group I. Hemodynamic parameters, arterial and mixed venous blood gases, and plasma thromboxane B2 and prostaglandin 6-keto-F1 were measured. At sacrifice after 4 hours, wet-to-dry lung weights were calculated. Results indicated that thromboxane A2 was necessary and sufficient for the development of pulmonary hypertension and impaired alveolar-capillary oxygen diffusion in graded bacteremia. It was necessary but not sufficient for increased lung water to occur and sufficient but not necessary for decreased cardiac index and stroke volume index. Thromboxane A2 was neither sufficient nor necessary to the pathophysiology of systemic hypotension during graded bacteremia. Plasma prostaglandin 6-keto-F1 levels were increased in hypotensive animals with sepsis, suggesting its involvement in hypotension during sepsis.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Aeromonas; Animals; Blood Pressure; Bridged Bicyclo Compounds, Heterocyclic; Capillary Permeability; Cardiac Output; Epoprostenol; Fatty Acids, Unsaturated; Hemodynamics; Hydrazines; Hypertension, Pulmonary; Hypoxia; Male; Prostaglandin Endoperoxides, Synthetic; Pulmonary Gas Exchange; Sepsis; Shock, Septic; Swine; Thromboxane A2; Thromboxane B2