Nitric oxide and cyclic guanosine monophosphate stimulate apoptosis via activation of the Fas-FasL pathway.

BACKGROUND: Inappropriately exaggerated response of pulmonary vascular cells to inflammatory mediators may be one mechanism that leads to acute (or adult) respiratory distress syndrome. Nitric oxide (NO) is induced following such exaggerated responses and may have a variety of biological effects, including induction of apoptosis. The mechanism by which NO causes apoptosis is unknown; however, Fas (CD95) and Fas ligand (FasL) (CD95L) have been implicated. We hypothesized that NO-induced apoptosis in pulmonary vascular smooth muscle cells is mediated through a Fas-FasL pathway. MATERIALS AND METHODS: Cultured human and rat pulmonary artery smooth muscle cells (PASMCs) were exposed to soluble FasL (0-5 ng/ml), the NO donor S(G)-nitroso-N-acetyl pencillamine (SNAP) (0-50 microg/ml), and/or anti-FasL (0-100 microg/ml) for 12 h. Apoptosis was measured using in situ DNA nick end labeling and flow cytometry. Changes in Fas and FasL protein levels were assessed via Western blot analysis. Messenger RNA (mRNA) abundance of apoptosis-related genes was determined using a ribonuclease protection assay. RESULTS: Rat PASMCs exposed to FasL show a dose-dependent increase in apoptosis. Human PASMCs are less responsive to FasL. Addition of anti-FasL to rat PASMCs treated with 10(-5) M SNAP decreases apoptosis levels compared to SNAP treated alone. FasL and Fas receptor proteins are increased in response to 10(-3) to 10(-4) M SNAP or 10(-6) M 8-bromo-cyclic guanosine monophosphate (cGMP). The mRNA abundance of Fas, FasL, and other apoptosis-related genes is increased in response to 10(-6) M 8-bromo-cGMP but not 8-bromo-cyclic adenosine monophosphate. CONCLUSIONS: Nitric oxide-induced apoptosis in rat and human PASMCs is mediated, at least in part, through the Fas-FasL pathway, with cGMP increasing the expression of Fas and FasL.

Cyclic nucleotides and inducible nitric oxide synthesis in pulmonary artery smooth muscle.

BACKGROUND: Nitric oxide (NO), cGMP, and cAMP affect the synthesis, metabolism, and cellular effects each other. We wanted to study how cGMP and cAMP interact to affect the induced synthesis of NO in response to interleukin-1 beta (IL-1 beta) in rat pulmonary artery smooth muscle cells. To further dissect the relative contributions of each cyclic nucleotide, and to detect any possible "crossover" effect of one cyclic nucleotide activating the other protein kinase, we tested how pharmacological inhibition of cGMP-dependent and cAMP-dependent protein kinases (PKG and PKA, respectively) affected responses. MATERIALS AND METHODS: We tested the effects of IL-1 beta, dibutyryl (db)-cAMP (1-100 micro) and 8-bromo (Br)-cGMP (1 microM-1 mM) on NO synthesis in cultured rat pulmonary artery smooth muscle cells. Positive effects were then tested in the presence of KT5720 (10(-9)-10(-5) M), the pharmacological inhibitor of PKA, and KT5823 (10(-9)-10(-5) M), the pharmacological inhibitor of PKG. NO production was measured using the Greiss reaction, and mRNA abundance of the inducible NO synthase (iNOS), using semiquantitative RT-PCR. RESULTS: IL-1 beta caused nitrite levels to increase nearly 10-fold over basal levels at 24 h (P < 0.05). Nitrite levels increased with the addition of either db-cAMP (100 microM, an 8-fold increase) or 8-Br-cGMP (100 microM, a 3-fold increase) to IL-1 beta (P < 0.05). PKA inhibition with KT5720 (10(-5) M) completely inhibited NO synthesis in response to the combination of IL-1 beta and cAMP, while KT5823 had less effect at all doses tested. NO synthesis in response to IL-1 beta plus cGMP also decreased to PKA inhibition, but not PKG inhibition, indicating that cGMP responses are a crossover effect. Both cAMP and cGMP in combination with IL-1 beta increased iNOS mRNA abundance above basal levels on reverse transcription polymerase chain reaction. KT5720, but not KT5823, decreased iNOS mRNA to basal levels. CONCLUSION: Both cAMP and cGMP augment cytokine induction of NO synthesis through activation of PKA: cAMP does so directly; cGMP, through a crossover stimulation of PKA.

Nitric oxide causes apoptosis in pulmonary vascular smooth muscle cells.

Nitric oxide (NO), a product of certain cytokine-activated cells, affects rates of apoptosis, a mechanism of programmed cell death. We asked whether NO affected rates of apoptosis in pulmonary vascular cells. Using rat pulmonary artery smooth muscle cells, we studied direct effects of the NO donor SG-nitroso-acetyl-D,L-penicillamine (SNAP) and the effects of NO endogenously synthesized in response to bacterial lipopolysaccharide (LPS) and inflammatory cytokines interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha (a combination called cytomix for convenience). We determined apoptosis on the basis of light microscopy and the bromodeoxyuridine terminal deoxynucleotidyl transferase reaction (BrdUTdT). Both SNAP- and cytomix-induced synthesis of NO resulted in histologic evidence of apoptosis based upon fluorescence microscopy using propidium iodide. SNAP (10(-5) M) increased BrdUTdT-positive cells from 17.5 to 78.4% compared with basal medium alone, with the maximal response occurring at 15 h or exposure. Exposing cells to LPS and cytokines induced NO production (from 0.1 +/- 0.1 to 24.6 +/- 0.5 microM, P < 0.05) caused cytological changes consistent with apoptosis and led to an increase of increased BrdUTdT-positive cells from 11 to 41% at 12 h compared with basal medium alone. The competitive NO synthase inhibitor NG-monomethyl-L-arginine inhibited both NO synthesis and NO apoptosis, returning the proportion of BrdUTdT-positive cells (6%) to levels below control. L-Arginine (0.5 mM) restored percentages to those increase in response to endogenously synthesized NO, and NO is a potential mechanism of acute lung injury in response to inflammatory cytokines.

Sustained nitric oxide exposure decreases soluble guanylate cyclase mRNA and enzyme activity in pulmonary artery smooth muscle.

BACKGROUND: The soluble isoform of guanylate cyclase (sGC) is activated by nitric oxide (NO) to form guanoside 3':5'-cyclic monophosphate (cGMP). Cyclic GMP levels cause smooth muscle relaxation and regulate vascular tone to various vascular beds, including the lung. Under conditions of cytokine excess the inducible synthesis of NO may result in cGMP overproduction, generalized vasodilatation, and septic shock. In the pulmonary bed the opposite response, pulmonary hypertension, may occur. We hypothesized that sGC activity decreases in the face of sustained levels of NO. MATERIALS AND METHODS: We used the NO-donor S-nitroso-acetyl-D-L-penicillamine to study the effects of NO on sGC mRNA abundance and enzyme activity in cultured rat pulmonary artery smooth muscle cells. RESULTS: NO caused a prompt rise in extracellular cGMP production. Pretreating cells with NO for >/=45 min inhibited subsequent cGMP synthesis. NO-pretreated cells recovered the capacity for cGMP synthesis after removal of NO for 120 min. When actinomycin or cycloheximide was added to NO pretreatment, cells retained cGMP synthetic capacity. NO pretreatment decreased sGC mRNA abundance, but did not totally eliminate it. CONCLUSION: NO has important regulatory effects on cGMP synthesis at the level of enzyme activity and mRNA abundance. NO causes an immediate synthesis of large amounts of cGMP. With prolongation of exposure (>/=60 min) sGC enzyme activity decreases and cGMP production drops significantly. Soluble GC mRNA abundance also decreases and may result in decreased responsiveness of cells to NO with regard to cGMP production.

Nitric oxide inhibits peroxide-mediated endothelial toxicity.

BACKGROUND: Oxidant molecules and nitric oxide (NO) have each been implicated as mediators of endothelial cell damage, but the biologic effect of these molecules acting in concert is incompletely understood. MATERIALS AND METHODS: We studied the effects of an NO donor, S-nitroso-acetyl-D,L-penicillamine (SNAP), in combination with the peroxidants tert-butyl hydroperoxide (TBH) and hydrogen peroxide (H2O2) on rabbit aortic endothelial cells in culture. Cell viability was assessed using Alamar blue, a nontoxic dye indicator of cell metabolism. Lipid peroxidation was assessed using a chemiluminescent single-photon counting technique. RESULTS: After 90 min exposure to test reagents, there was concentration-dependent cytotoxicity for both TBH and H2O2. Peroxidant-induced cytotoxicity was significantly ameliorated by SNAP (10(-4)-10(-3)M). N-Acetylpenicillamine and NO-depleted SNAP failed to demonstrate a cytoprotective effect against peroxidant cellular injury, thus implicating NO as the agent responsible for the protective effect. SNAP reduced lipid peroxidation caused by 10(-3) M TBH in a dose-dependent manner. Preincubation of cells with SNAP before exposure to peroxidants alone had no effect on toxicity. CONCLUSIONS: NO is cytoprotective to the endothelium in the presence of peroxidants through a reduction of lipid peroxidation.

Escherichia coli lipopolysaccharide downregulates soluble guanylate cyclase in pulmonary artery smooth muscle.

The soluble isoform of guanylate cyclase (sGC) is activated by nitric oxide (NO) to form guanosine 3':5'-cyclic monophosphate (cGMP). Cyclic GMP levels cause smooth muscle relaxation and regulate vascular tone to various vascular beds, including the lung. Under conditions of cytokine excess the inducible synthesis of NO may result in cGMP overproduction, generalized vasodilation, and septic shock. In the pulmonary bed the opposite response may occur, pulmonary hypertension. We hypothesized that sGC activity becomes downregulated in the face of Escherichia coli lipopolysaccharide (LPS). We tested the effects of LPS on alpha1-subunit sGC mRNA abundance, Western analysis, and enzyme activity in cultured rat pulmonary artery smooth muscle cells. LPS increased extracellular cGMP production by pulmonary artery smooth muscle cells, with increased levels being first detectable at 3-6 h (10 microg/ml LPS) and exceeding 140 pmol/ml by 24 h (P < 0.05). The response was inhibited by 0.05 mM l-NG-monomethyl-l-arginine (l-NMA) and, in turn, restored by 1 mM l-arginine, indicating a NO synthase-dependent response. Pretreating cells with LPS for >/= 3 h inhibited subsequent cGMP synthesis in response to 10(-4) M SNAP for 60 min. Coincubating cells with 0.05 mM l-NMA also reversed this effect. Soluble GC enzyme activity in cells exposed to basal medium alone measured 0.74 pmol cGMP/ml per minute; activity in cells exposed to 10 microg/ml LPS for 24 h decreased to 0.04 pmol cGMP/ml per minute (P < 0.05). LPS pretreatment decreased sGC mRNA abundance and protein mass, but did not totally eliminate them. It is concluded that LPS affects cGMP synthesis at the level of enzyme activity, enzyme mass, and mRNA abundance. Over the short term (<24 h) LPS causes the synthesis of large amounts of cGMP. As the duration of exposure progresses (>/=3 h), mechanisms come into play that decrease cGMP production significantly and include decreases in mRNA abundance, enzyme mass, and enzyme activity.

Efficacy of inhaled nitric oxide in oleic acid-induced acute lung injury.

OBJECTIVE: To assess the efficacy of inhaled nitric oxide in improving pulmonary hypertension and gas exchange following oleic acid-induced acute lung injury. DESIGN: Prospective, pharmacologic study. SETTING: Surgical research laboratory at the University of Pittsburgh, Pittsburgh, PA. SUBJECTS: Instrumented, intubated pigs weighing 16 to 27 kg. INTERVENTIONS: Intravenous oleic acid and inhaled nitric oxide. MEASUREMENTS AND MAIN RESULTS: All pigs treated with intravenous oleic acid (0.11 mL/kg) developed a severe lung injury with pulmonary hypertension, accompanied by impaired oxygenation, intrapulmonary shunting, and increased extravascular lung water (p < .05 compared with baseline). Following nitric oxide inhalation, although pulmonary hypertension decreased in a dose-dependent fashion, no amelioration in pulmonary gas exchange was observed, as reflected by PaO2 and intrapulmonary shunt. Plasma nitrite and nitrate concentrations, the stable end products of nitric oxide metabolism, did not increase following nitric oxide exposure in this model of severe lung injury. CONCLUSIONS: The effect of inhaled nitric oxide, restricted to relieving pulmonary vasoconstriction in this model of lung injury, may have limited benefit in improving pulmonary gas exchange when diffusion is impaired by severe lung injury and inflammatory thickening of the alveolar-capillary barrier. Nitric oxide inhalation may have better results when used at an earlier, less severe stage of acute lung injury.

Nitric oxide and the pulmonary artery smooth muscle cell.

Nitric oxide (NO) is produced by the enzyme nitric oxide synthase (NOS), which exists in different isoforms in various tissues. The inducible NOS (iNOS) isoform of the enzyme is expressed in vascular smooth muscle in response to lipopolysaccharide and inflammatory mediators. When this expression of iNOS occurs in the lung, the NO produced may play a role in the inflammatory process of acute lung injury. This article reviews the research that characterizes iNOS in rat pulmonary artery smooth muscle and discusses current investigation into the role of NO in sepsis and injury.

Perinatal management of a lingual teratoma.

BACKGROUND: Teratomas of the head and neck are rare and occur almost exclusively in neonates. Prenatal diagnosis of these tumors allows for a carefully planned delivery that maintains an open airway and potentially improves perinatal outcome. We report the perinatal management of a huge intrapharyngeal and intra-oral teratoma that had a broad connection to the base of the tongue. CASE: An anterior neck mass (5 x 5 cm) in an otherwise normal-appearing fetus was detected at 19 weeks' gestation. The mass increased in size over the next 10 weeks to 8 x 6.8 x 4.3 cm. Marked fetal head deflexion was noted along with concomitant hydramnios. The mother had spontaneous rupture of membranes with preterm labor at 29 weeks' gestation. A 1860-g male neonate was delivered by classical cesarean delivery. A tracheostomy was performed in the delivery room for ventilation after the upper airway could not be accessed by bronchoscopy. Histologic examination after surgical excision confirmed a congenital teratoma with immature neuroectodermal tissue and alpha-fetoprotein-bearing endodermal sinus tumor components with exclusively polyvesicular vitelline characteristics. The origin of the pharyngeal mass was the base of the tongue. CONCLUSION: The prenatal diagnosis of a pharyngeal teratoma should prompt a careful delivery plan to optimize perinatal outcome.

Etiology and outcome of pediatric burns.

A 6-year retrospective review of burn victims hospitalized at a major burn center was conducted to determine the etiology and outcome of pediatric burns. Four hundred forty-nine patients under age 16 years were identified and stratified by age, sex, burn size, presence or absence of inhalation injury, cause of burn, and county of residence. The mean patient age was 4.3 +/- 0.2 years, and the male:female ratio was 1.9:1. There were 21 deaths overall (4.7%), the majority of which (18) were among children under 4 years of age. With respect to large burns, defined as > and = 30% total body surface area (TBSA), the mortality rate for children under age 4 was significantly higher than that for older children (46.9% v 12.5%; P < .01), despite the nearly identical mean burn size of the two groups. Except for burn incidence, there were no significant differences between males and females. The mean burn size was 15.1% +/- 0.7%, and was significantly larger for nonsurvivors than survivors (55.3% +/- 5.7 v 13.1% +/- 0.5%; P < .01). Inhalation injuries were strongly associated with large burns and were present in all 15 flame-burn fatalities. Scalds were the most common type of burn among children under 4 years of age; flame burns predominated in older children. There were 6 deaths related to scalds, all of which occurred in children under 4. Burn type, size, and mortality rate did not differ between children from urban and rural counties. Large burn size was the strongest predictor of mortality, followed by (in order) age less than 4 and the presence of inhalation injury. Infants and young children have the highest risk of death from burn injury. Burns smaller than 30% TBSA without an inhalation injury (such as small scald injuries) occasionally are lethal in infants and small children, despite modern therapy.

Nitric oxide produced by cytokine-activated pulmonary artery smooth muscle cells is cytotoxic to cocultured endothelium.

BACKGROUND: We recently demonstrated that rat pulmonary artery smooth muscle (RPASM) generates maximal nitric oxide (NO) when exposed to inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. Our hypothesis is that NO produced by cytokine-stimulated RPASM has local cytotoxic effects on endothelium. Accordingly, we designed a pulmonary smooth muscle and endothelial coculture experiment in which the effects of NO on endothelium can be distinguished from the direct effects of cytokines. METHODS: RPASM cells were incubated with a mixture of TNF-alpha (500 units/ml) and IFN-gamma (100 units/ml) for 24 hours. This cytokine mixture was then removed and the NO-producing smooth muscle cells were incubated in a coculture transwell system with rat pulmonary artery endothelial (RPAE) cells. Subsequent NO production (as measured by nitrite concentration in cell supernatants), and the number of viable attached endothelial cells were then measured at 48 hours. RESULTS: RPASM continued to produce large amounts of NO, in the absence of further cytokine stimulation, after a 24-hour exposure to TNF-alpha and IFN-gamma. This RPASM-generated NO decreased the number of viable attached endothelial cells after 24 hour RPASM-RPAE coculture by 57%. The competitive stereospecific inhibitor of inducible NO synthase (iNOS), NG-monomethyl-L-arginine (NMA), returned the inducible NO production to basal levels and reversed the cytotoxic effects on endothelial cells. The number of viable attached endothelial cells returned to control levels. CONCLUSIONS: The NO produced by cytokine-activated RPASM has local cytotoxic effects on RPAE in coculture. Such NO produced in the vasculature may be a factor in the origin of acute lung injury under conditions of trauma and sepsis.

The effect of nitric oxide on fetal pulmonary artery smooth muscle growth.

Endothelium-derived nitric oxide (NO) relaxes fetal pulmonary arterial vessels through activation of guanylate cyclase and increasing smooth muscle cyclic guanosine 3', 5'-monophosphate (cGMP). Exogenous NO administered as a gas at low concentrations shares this effect, decreasing pulmonary artery resistance and increasing in pulmonary blood flow. NO, endogenously synthesized or inhaled as a gas, may affect cellular growth in the underlying pulmonary vascular smooth muscle media. We report the effects of NO and cGMP upon DNA synthesis and proliferation of passaged pulmonary vascular smooth muscle cells from fetal rats. Smooth muscle cells from rat fetal pulmonary artery (RFPASM; 18-19 day gestation; term 21 days) were treated in culture with sodium nitroprusside (SNP), isosorbide dinitrite (ISDN)--both NO-generating vasodilators--or 8-bromo-cGMP, a cell-permeant cGMP analog. All agents inhibited thymidine uptake at concentrations of 10(-3)-10(-2) M. Lower concentrations (10(-5)-10(-4) M) of SNP and ISDN increased [3H]-thymidine ([3H]TdR) uptake, an effect not seen with cGMP at similar concentrations. Exposing RFPASM to authentic NO gas in a deoxygenated medium inhibited [3H]TdR uptake only. NO appears to have a biphasic effect on DNA synthesis in passaged RFPASM, with stimulation at micromolar concentrations and inhibition at higher levels. NO may thus alter vascular smooth muscle growth and pulmonary vascular remodeling in conditions complicated by pulmonary hypertension and treated with inhaled NO.

Prenatal diagnosis and management of massive bilateral axillary cystic lymphangioma.

BACKGROUND: Fetal lymphangiomas can occur in many different anatomic locations, including the most commonly seen nuchal cystic hygroma. CASE: A fetus at 18 weeks' gestation was found to have a massive right axillary hygroma. The fetal karyotype was normal. Serial ultrasound examinations indicated progressive enlargement, but no hydrops. At 32 weeks' gestation, a left axillary hygroma was also diagnosed. The patient underwent cesarean delivery. CONCLUSION: Prenatal diagnosis of nuchal cystic hygromas has a high association with karyotypic abnormalities, hydrops, and fetal demise; however, this association may not apply to cystic lymphangiomas at other locations.

TGF-beta regulates production of NO in pulmonary artery smooth muscle cells by inhibiting expression of NOS.

We have previously reported that a mixture of lipopolysaccharide and cytokines stimulates cultured rat pulmonary artery smooth muscle cells (RPASM) to express elevated levels of mRNA for inducible nitric oxide synthase (iNOS), and to produce large amounts of nitric oxide (NO). The current study tests the hypothesis that transforming growth factor-beta (TGF-beta) modulates this process. Accordingly, RPASM were treated with a mixture of LPS (10 micrograms/ml) and the cytokines interleukin-1 beta (5 U/ml), tumor necrosis factor-alpha (500 U/ml), and interferon-gamma (100 U/ml). In the absence of TGF-beta 1, NO production (indicated by colorimetric assay of cumulative nitrite levels at 24 h) was greatly increased, as previously observed. Under identical conditions, TGF-beta 1 caused a concentration-dependent decrease in NO production. The addition of neither excess L-arginine nor sepiapterin reversed the inhibition, indicating that the effect of TGF-beta 1 was not due to limitation of enzyme substrate or cofactor tetrahydrobiopterin, respectively. Northern and Western analyses showed that TGF-beta 1 reduced levels of iNOS mRNA and protein to baseline at all time points examined up to 24 h. Complete suppression of iNOS protein expression was evident even when TGF-beta 1 was added at postinduction time points. One mechanism of action of TGF-beta 1 was demonstrated in experiments in which degradation of iNOS protein was greatly increased by the addition of TGF-beta 1. These results demonstrate that TGF-beta 1 regulates production of NO in RPASM by inhibiting iNOS expression in part by increasing degradation of existing iNOS protein.

Culture of pulmonary microvascular smooth muscle cells from intraacinar arteries of the rat: characterization and inducible production of nitric oxide.

Pulmonary arterial microvascular smooth muscle function governs many aspects of lung physiology and pathophysiology. Acutely, microvascular smooth muscle cells (SMC) modulate pulmonary vascular resistance; chronically, they contribute to vascular remodeling. Recent work has also suggested a possible immune function for pulmonary smooth muscle through cytokine-stimulated nitric oxide production. To facilitate study of the mechanisms underlying these functions, we have developed methods for isolating pulmonary arterial microvessels from the rat and culturing SMC from these vessels. The pulmonary arterial circulation was filled with a suspension of iron oxide in agar, and a subpleural tissue sample was obtained. The vessels were cleared of surrounding lung parenchyma by partial collagenase digestion, and the iron-containing arteries were separated magnetically. The diameter of the harvested arteries confirmed an intraacinar origin, and the cultured cells expressed smooth muscle isoforms of alpha-actin and myosin but did not take up acetylated low density lipoprotein. To assess a possible immune effector role for these cells, confluent monolayers were stimulated with cytokines and endotoxin. At 24 h, immunofluorescent staining for inducible nitric oxide synthase was prominent within these cells. Nitric oxide production, as measured by nitrite levels in the cell-conditioned medium, was also markedly elevated but reduced by adding NG-monomethyl-L-arginine. We conclude that rat pulmonary arterial microvascular SMC can be obtained by the iron oxide infusion method and that these cells express an inducible nitric oxide synthase after cytokine stimulation.

Hemodynamic effects and metabolic fate of inhaled nitric oxide in hypoxic piglets.

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

Tetrahydrobiopterin synthesis and inducible nitric oxide production in pulmonary artery smooth muscle.

We recently reported (Am. J. Respir. Cell Mol. Biol. 7: 471-476, 1992) that a mixture of lipopolysaccharide (LPS) and cytokines produced a time-dependent increase in mRNA and protein expression of inducible nitric oxide synthase (iNOS) in cultured rat pulmonary artery smooth muscle cells (RPASM). In the current study we extend observations on regulation of iNOS in RPASM by showing that de novo synthesis of tetrahydrobiopterin (BH4) is critical for LPS and cytokine-induced NO production. A mixture of LPS and the cytokines gamma-interferon, interleukin-1 beta, and tumor necrosis factor-alpha increased steady-state levels of mRNA of GTP-cyclohydrolase-I (GTP-CH), the rate-limiting enzyme in BH4 biosynthesis. Levels of mRNA to GTP-CH became detectable by 4 h, with further increases at 24 h by Northern blot analysis and reverse-transcriptase polymerase chain reaction. Total intracellular biopterin levels, undetectable under basal conditions, increased after 24 h exposure to LPS and cytokines (to 32.3 +/- 0.8 pmol/mg protein). LPS and cytokine-induced NO production, determined by nitrite concentrations in the medium, was decreased in a concentration-dependent manner by the GTP-CH inhibitor, 2,4-diamino-6-hydroxypyrimidine (DAHP) at 24 h. DAHP also inhibited completely the LPS- and cytokine-induced accumulation of intracellular biopterins. Sepiapterin, which supplies BH4 through a salvage pathway independent of GTP-CH, reversed the effect of DAHP on LPS and cytokine-induced NO production.(ABSTRACT TRUNCATED AT 250 WORDS)