Decreased association of HSP90 impairs endothelial nitric oxide synthase in fetal lambs with persistent pulmonary hypertension

Persistent pulmonary hypertension of newborn (PPHN) is associated with decreased nitric oxide (NO) release and impaired pulmonary vasodilation. We investigated the hypothesis that decreased association of heat shock protein 90 (HSP90) with endothelial NO synthase (eNOS) impairs NO release and vasodilation in PPHN. The responses to the NOS agonist ATP were investigated in fetal lambs with PPHN induced by prenatal ligation of ductus arteriosus, and in sham ligation controls. ATP caused dose-dependent vasodilation in control pulmonary resistance arteries, and this response was attenuated in PPHN vessels. The response of control pulmonary arteries to ATP was attenuated by NG-nitro-l-arginine methyl ester (l-NAME), a NOS antagonist, and geldanamycin, an inhibitor of HSP90-eNOS interaction. The attenuated response to ATP observed in PPHN was improved by pretreatment of vessels with l-NAME or 4,5-dihydroxy-1,3-benzene-disulfonate, a superoxide scavenger. Pulmonary arteries from PPHN lambs had decreased basal levels of HSP90 in association with eNOS. Association of HSP90 with eNOS and NO release increased in response to ATP in control pulmonary artery endothelial cells, but not in cells from PPHN lambs. Decreased HSP90-eNOS interactions may contribute to the impaired NO release and vasodilation observed in the ductal ligation model of PPHN.     

K+ channel pulmonary vasodilation in fetal lambs: role of endothelium-derived nitric oxide.

To define the role and mechanism of action of K+ channels in regulating fetal pulmonary vascular tone, we studied the hemodynamic effects of pinacidil (a K+ channel activator) and glibenclamide (a K+ channel blocker). The effects of pinacidil were compared with those of acetylcholine [an endothelium-derived relaxing factor- (EDRF) dependent pulmonary vasodilator] and 8-bromoguanosine 3',5'-cyclic monophosphate (8-bromo-cGMP, an EDRF-independent pulmonary vasodilator) before and after treatment with N omega-nitro-L-arginine [a competitive inhibitor of an EDRF, endothelium-derived nitric oxide (EDNO), synthesis], or L-arginine (the substrate for the formation of EDNO). In 14 unanesthetized fetal lambs in utero, catheters were inserted into the fetal pulmonary artery, descending aorta, left atrium, and superior vena cava to measure pressures and administer drugs. An ultrasonic flow transducer was placed around the left pulmonary artery to measure flow (QP) continuously. In eight animals, pinacidil, acetylcholine, and 8-bromo-cGMP caused similar acute maximal increases in QP of 128, 137, and 155 ml/min, respectively. After a 60-min infusion of N omega-nitro-L-arginine (2.07 +/- 0.27 mg.kg-1.min-1), the increase in QP caused by acetylcholine and pinacidil was significantly attenuated, by 84 and 68%, respectively, with only a 10% attenuation of the increase in QP caused by 8-bromo-cGMP. In six additional N omega-nitro-L-arginine-pretreated fetal lambs, infusion of L-arginine (32.2 +/- 4.3 mg.kg-1.min-1) restored the vasodilatory effects of acetylcholine and pinacidil. A 20-min infusion of glibenclamide (n = 6; 0.64 +/- 0.07 mg.kg-1.min-1) blocked the vasodilation by pinacidil but not acetylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective pulmonary vasodilation with ATP-MgCl2 during pulmonary hypertension in lambs

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.     

Decreased synthesis and vasodilation to nitric oxide in piglets with hypoxia-induced pulmonary hypertension

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

Upregulation of nitric oxide synthase in mice with severe hypoxia-induced pulmonary hypertension

Background  

The importance of nitric oxide (NO) in hypoxic pulmonary hypertension has been demonstrated using nitric oxide synthase (NOS) knockout mice. In that model NO from endothelial NOS (eNOS) plays a central role in modulating pulmonary vascular tone and attenuating hypoxic pulmonary hypertension. However, the normal regulation of NOS expression in mice following hypoxia is uncertain. Because genetically engineered mice are often utilized in studies of NO, we conducted the present study to determine how hypoxia alters NOS expression in wild-type mice.     

Pulmonary vasodilation by nitric oxide gas and prodrug aerosols in acute pulmonary hypertension

Adrie, Christophe, Fumito Ichinose, AlexandraHolzmann, Larry Keefer, William E. Hurford, and Warren M. Zapol. Pulmonary vasodilation by nitric oxide gas and prodrugaerosols in acute pulmonary hypertension. J. Appl. Physiol. 84(2): 435-441, 1998.Sodium 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate{DEA/NO;Et₂N[N(O)NO]Na} is a compound that spontaneously generates nitric oxide (NO). Becauseof its short half-life (2.1 min), we hypothesized that inhaling DEA/NOaerosol would selectively dilate the pulmonary circulation withoutdecreasing systemic arterial pressure. We compared the pulmonaryselectivity of this new NO donor with two other reference drugs:inhaled NO and inhaled sodium nitroprusside (SNP). In seven awake sheepwith pulmonary hypertension induced by the infusion of U-46619, wecompared the hemodynamic effects of DEA/NO with those of incrementaldoses of inhaled NO gas. In seven additional awake sheep, we examinedthe hemodynamic effects of incremental doses of inhaled nitroprusside(i.e., SNP). Inhaled NO gas selectively dilated the pulmonaryvasculature. Inhaled DEA/NO produced nonselective vasodilation; bothsystemic vascular resistance (SVR) and pulmonary vascular resistance(PVR) were reduced. Inhaled SNP selectively dilated the pulmonarycirculation at low concentrations(10² M), inducing adecrease of PVR of up to 42% without any significant decrease of SVR(5%), but nonselectively dilated the systemic circulation atlarger doses (>10² M). Inconclusion, despite its short half-life, DEA/NO is not a selectivepulmonary vasodilator compared with inhaled NO. Inhaled SNP appears tobe selective to the pulmonary circulation at low doses but not athigher levels.

     

Deficiency in endothelial nitric oxide synthase impairs myocardial angiogenesis

We recently demonstrated that mice deficient in endothelial nitric oxide (NO) synthase (eNOS) have congenital septal defects and postnatal heart failure. However, the mechanisms by which eNOS affects heart development are not clear. We hypothesized that deficiency in eNOS impairs myocardial angiogenesis. Myocardial capillary densities were measured morphometrically in neonatal mouse hearts. In vitro tube formation on Matrigel was investigated in cardiac endothelial cells. In vivo myocardial angiogenesis was performed by implanting Matrigel in the left ventricular myocardium. Myocardial capillary densities and VEGF mRNA expression were decreased in neonatal eNOS(-/-) compared with neonatal wild-type mice (P < 0.01). Furthermore, in vitro tube formation from cardiac endothelial cells and in vivo myocardial angiogenesis were attenuated in eNOS(-/-) compared with wild-type mice (P < 0.01). In vitro tube formation was inhibited by N(G)-nitro-l-arginine methyl ester in wild-type mice and restored by a NO donor, diethylenetriamine-NO, in eNOS(-/-) mice (P < 0.05). In conclusion, deficiency in eNOS decreases VEGF expression and impairs myocardial angiogenesis and capillary development. Decreased myocardial angiogenesis may contribute to cardiac abnormalities during heart development in eNOS(-/-) mice.     

Cell growth modulates nitric oxide synthase expression in fetal pulmonary artery endothelial cells

Nitric oxide (NO), produced by endothelial (e) nitric oxide synthase (NOS), is a critical mediator of vascular function and growth in the developing lung. Pulmonary eNOS expression is diminished in conditions associated with altered pulmonary vascular development, suggesting that eNOS may be modulated by changes in pulmonary artery endothelial cell (PAEC) growth. We determined the effects of cell growth on eNOS expression in cultured ovine fetal PAEC studied at varying levels of confluence. NOS enzymatic activity was sixfold greater in quiescent PAEC at 100% confluence compared with more rapidly replicating cells at 50% confluence. To determine if there is a reciprocal effect of NO on PAEC growth, studies of NOS inhibition or the provision of exogenous NO from spermine NONOate were performed. Neither intervention had a discernable effect on PAEC growth. The influence of cell growth on NOS activity was unique to pulmonary endothelium, because varying confluence did not alter NOS activity in fetal systemic endothelial cells. The effects of cell growth induced by serum stimulation were also evaluated, and NOS enzymatic activity was threefold greater in quiescent, serum-deprived cells compared with that in serum-stimulated cells. The increase in NOS activity observed at full confluence was accompanied by parallel increases in eNOS protein and mRNA expression. These findings indicate that eNOS gene expression in fetal PAEC is upregulated during cell quiescence and downregulated during rapid cell growth. Furthermore, the interaction between cell growth and NO in the PAEC is unidirectional.     

Inhibition of inducible nitric oxide synthase in persistent pain

The present study was undertaken to investigate the role of inducible nitric oxide synthase in a rat model of persistent pain. The effects of L-N6 (1-iminoethyl) lysine (L-NIL), a relatively potent and relatively selective inhibitor of inducible nitric oxide synthase, were investigated in carrageenan induced hyperalgesia L-NIL (0.1 microMole) injected intraplantar or intrathecal markedly enhanced carrageenan induced hyperalgesia. These effects were reversed during the third hour by co-administration of L-arginine (900 mg/kg i.p.) but not D-arginine. Methylene blue (MB), a soluble guanylate cyclase inhibitor, administered intrathecally (0.1 microg) had no effect on L-NIL potentiation of carrageenan hyperalgesia but abolished antinociception induced by L-arginine. Obtained results suggest that nitric oxide derived from inducible nitric oxide synthase play an inhibitory role in carrageenan produced hyperalgesia in rat.     

Bradykinin produces pulmonary vasodilation in fetal lambs: role of prostaglandin production

Bradykinin produces pulmonary vasodilation and also stimulates production of other pulmonary vasodilators, including prostaglandin I2 (PGI2) and endothelial-derived relaxing factor. In 12 chronically instrumented fetal lambs, we therefore investigated potential mediation of the bradykinin response by PGI2 or other cyclooxygenase products. A 15-min infusion of bradykinin (approximately 1 microgram/kg estimated fetal wt/min) increased fetal pulmonary blood flow by 522% (P less than 0.05) and decreased pulmonary vascular resistance by 86% (P less than 0.05); plasma 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) concentration also increased (P less than 0.05). After cyclooxygenase inhibition by indomethacin (3 mg), bradykinin increased pulmonary blood flow by only 350% (P less than 0.05) and decreased pulmonary vascular resistance by 83% (P less than 0.05); plasma 6-keto-PGF1 alpha concentrations did not increase. The increase in pulmonary blood flow produced by bradykinin was greater before administration of indomethacin than after (P less than 0.05). These studies demonstrate that bradykinin produces fetal pulmonary vasodilation by at least two mechanisms, one dependent on and the other independent of PGI2 production, the latter mechanism predominating.     

Inhibition of phosphodiesterase 1 augments the pulmonary vasodilator response to inhaled nitric oxide in awake lambs with acute pulmonary hypertension

Phosphodiesterase 1 (PDE1) modulates vascular tone and the development of tolerance to nitric oxide (NO)-releasing drugs in the systemic circulation. Any role of PDE1 in the pulmonary circulation remains largely uncertain. We measured the expression of genes encoding PDE1 isozymes in the pulmonary vasculature and examined whether or not selective inhibition of PDE1 by vinpocetine attenuates pulmonary hypertension and augments the pulmonary vasodilator response to inhaled NO in lambs. Using RT-PCR, we detected PDE1A, PDE1B, and PDE1C mRNAs in pulmonary arteries and veins isolated from healthy lambs. In 13 lambs, the thromboxane A(2) analog U-46619 was infused intravenously to increase mean pulmonary arterial pressure to 35 mmHg. Four animals received an intravenous infusion of vinpocetine at incremental doses of 0.3, 1, and 3 mg.kg(-1).h(-1). In nine lambs, inhaled NO was administered in a random order at 2, 5, 10, and 20 ppm before and after an intravenous infusion of 1 mg.kg(-1).h(-1) vinpocetine. Administration of vinpocetine did not alter pulmonary and systemic hemodynamics or transpulmonary cGMP or cAMP release. Inhaled NO selectively reduced mean pulmonary arterial pressure, pulmonary capillary pressure, and pulmonary vascular resistance index, while increasing transpulmonary cGMP release. The addition of vinpocetine enhanced pulmonary vasodilation and transpulmonary cGMP release induced by NO breathing without causing systemic vasodilation but did not prolong the duration of pulmonary vasodilation after NO inhalation was discontinued. Our findings demonstrate that selective inhibition of PDE1 augments the therapeutic efficacy of inhaled NO in an ovine model of acute chemically induced pulmonary hypertension.     

Superoxide dismutase restores eNOS expression and function in resistance pulmonary arteries from neonatal lambs with persistent pulmonary hypertension

Endothelial nitric oxide (NO) synthase (eNOS) expression and activity are decreased in fetal lambs with persistent pulmonary hypertension (PPHN). We sought to determine the impact of mechanical ventilation with O(2) with or without inhaled NO (iNO) or recombinant human SOD (rhSOD) on eNOS in the ductal ligation model of PPHN. PPHN lambs and age-matched controls were ventilated with 100% O(2) for 24 h alone or combined with 20 ppm iNO continuously or a single dose of rhSOD (5 mg/kg) given intratracheally at delivery. In 1-day spontaneously breathing lambs, eNOS expression in resistance pulmonary arteries increased relative to fetal levels. eNOS expression increased in control lambs ventilated with 100% O(2), but not in PPHN lambs. Addition of iNO or rhSOD increased eNOS expression and decreased generation of reactive oxygen species (ROS) in PPHN lambs relative to those ventilated with 100% O(2) alone. However, only rhSOD restored eNOS function, increased tetrahydrobiopterin (BH(4)), a critical cofactor for eNOS function, and restored GTP cyclohydrolase I expression in isolated vessels and lungs from PPHN lambs. These data suggest that ventilation of PPHN lambs with 100% O(2) increases ROS production, blunts postnatal increases in eNOS expression, and decreases available BH(4) in PPHN lambs. Although the addition of iNO or rhSOD diminished ROS production and increased eNOS expression, only rhSOD improved eNOS function and levels of available BH(4). Thus therapies designed to decrease oxidative stress and restore eNOS coupling, such as rhSOD, may prove useful in the treatment of PPHN in newborn infants.     

Altered carnitine homeostasis is associated with decreased mitochondrial function and altered nitric oxide signaling in lambs with pulmonary hypertension

Utilizing aortopulmonary vascular graft placement in the fetal lamb, we have developed a model (shunt) of pulmonary hypertension that mimics congenital heart disease with increased pulmonary blood flow. Our previous studies have identified a progressive development of endothelial dysfunction in shunt lambs that is dependent, at least in part, on decreased nitric oxide (NO) signaling. The purpose of this study was to evaluate the possible role of a disruption in carnitine metabolism in shunt lambs and to determine the effect on NO signaling. Our data indicate that at 2 wk of age, shunt lambs have significantly reduced expression (P < 0.05) of the key enzymes in carnitine metabolism: carnitine palmitoyltransferases 1 and 2 as well as carnitine acetyltransferase (CrAT). In addition, we found that CrAT activity was inhibited due to increased nitration. Furthermore, free carnitine levels were significantly decreased whereas acylcarnitine levels were significantly higher in shunt lambs (P < 0.05). We also found that alterations in carnitine metabolism resulted in mitochondrial dysfunction, since shunt lambs had significantly decreased pyruvate, increased lactate, and a reduced pyruvate/lactate ratio. In pulmonary arterial endothelial cells cultured from juvenile lambs, we found that mild uncoupling of the mitochondria led to a decrease in cellular ATP levels and a reduction in both endothelial NO synthase-heat shock protein 90 (eNOS-HSP90) interactions and NO signaling. Similarly, in shunt lambs we found a loss of eNOS-HSP90 interactions that correlated with a progressive decrease in NO signaling. Our data suggest that mitochondrial dysfunction may play a role in the development of endothelial dysfunction and pulmonary hypertension and increased pulmonary blood flow.     

Sepiapterin improves angiogenesis of pulmonary artery endothelial cells with in utero pulmonary hypertension by recoupling endothelial nitric oxide synthase

Persistent pulmonary hypertension of the newborn (PPHN) is associated with decreased blood vessel density that contributes to increased pulmonary vascular resistance. Previous studies showed that uncoupled endothelial nitric oxide (NO) synthase (eNOS) activity and increased NADPH oxidase activity resulted in marked decreases in NO bioavailability and impaired angiogenesis in PPHN. In the present study, we hypothesize that loss of tetrahydrobiopterin (BH4), a critical cofactor for eNOS, induces uncoupled eNOS activity and impairs angiogenesis in PPHN. Pulmonary artery endothelial cells (PAEC) isolated from fetal lambs with PPHN (HTFL-PAEC) or control lambs (NFL-PAEC) were used to investigate the cellular mechanisms impairing angiogenesis in PPHN. Cellular mechanisms were examined with respect to BH4 levels, GTP-cyclohydrolase-1 (GCH-1) expression, eNOS dimer formation, and eNOS-heat shock protein 90 (hsp90) interactions under basal conditions and after sepiapterin (Sep) supplementation. Cellular levels of BH4, GCH-1 expression, and eNOS dimer formation were decreased in HTFL-PAEC compared with NFL-PAEC. Sep supplementation decreased apoptosis and increased in vitro angiogenesis in HTFL-PAEC and ex vivo pulmonary artery sprouting angiogenesis. Sep also increased cellular BH4 content, NO production, eNOS dimer formation, and eNOS-hsp90 association and decreased the superoxide formation in HTFL-PAEC. These data demonstrate that Sep improves NO production and angiogenic potential of HTFL-PAEC by recoupling eNOS activity. Increasing BH4 levels via Sep supplementation may be an important therapy for improving eNOS function and restoring angiogenesis in PPHN.     

Asymmetric dimethylarginine, oxidative stress, and vascular nitric oxide synthase in essential hypertension

We reported impaired endothelium-derived relaxation factor/nitric oxide (EDRF/NO) responses and constitutive nitric oxide synthase (cNOS) activity in subcutaneous vessels dissected from patients with essential hypertension (n = 9) compared with normal controls (n = 10). We now test the hypothesis that the patients in this study have increased circulating levels of the cNOS inhibitor, asymmetric dimethylarginine (ADMA), or the lipid peroxidation product of linoleic acid, 13-hydroxyoctadecadienoic acid (HODE), which is a marker of reactive oxygen species. Patients had significantly (P < 0.001) elevated (means +/- SD) plasma levels of ADMA (P(ADMA), 766 +/- 217 vs. 393 +/- 57 nmol/l) and symmetric dimethylarginine (P(SDMA): 644 +/- 140 vs. 399 +/- 70 nmol/l) but similar levels of L-arginine accompanied by significantly (P < 0.015) increased rates of renal ADMA excretion (21 +/- 9 vs. 14 +/- 5 nmol/mumol creatinine) and decreased rates of renal ADMA clearance (18 +/- 3 vs. 28 +/- 5 ml/min). They had significantly increased plasma levels of HODE (P(HODE): 309 +/- 30 vs. 226 +/- 24 nmol/l) and renal HODE excretion (433 +/- 93 vs. 299 +/- 67 nmol/micromol creatinine). For the combined group of normal and hypertensive subjects, the individual values for plasma levels of ADMA and HODE were both significantly (P < 0.001) and inversely correlated with microvascular EDRF/NO and positively correlated with mean blood pressure. In conclusion, elevated levels of ADMA and oxidative stress in a group of hypertensive patients could contribute to the associated microvascular endothelial dysfunction and elevated blood pressure.     

Nitric oxide synthase in pulmonary hypertension: lessons from knockout mice

Nitric oxide (NO) is implicated in a wide variety of biological roles. NO is generated from three nitric oxide synthase (NOS) isoforms: neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) all of which are found in the lung. While there are no isoform-specific inhibitors of NOS, the recent development and characterization of mice deficient in each of the NOS isoforms has allowed for more comprehensive study of the importance of NO in the lung circulation. Studies in the mouse have identified the role of NO from eNOS in modulating pulmonary vascular tone and in attenuating the development of chronic hypoxic pulmonary hypertension.     

Reduced endothelial nitric oxide synthase in lungs of chronically ventilated preterm lambs

Nitric oxide (NO), produced in lung vascular endothelium and airway epithelium, has an important role in regulating smooth muscle cell growth and tone. Chronic lung disease, a frequent complication of premature birth, is characterized by excess abundance, tone, and reactivity of smooth muscle in the pulmonary circulation and conducting airways, leading to increased lung vascular and airway resistance. Whether these structural and functional changes are associated with diminished pulmonary expression of endothelial nitric oxide synthase (eNOS) protein is unknown. Both quantitative immunoblot analysis and semiquantitative immunohistochemistry showed that there was less eNOS protein in the endothelium of small intrapulmonary arteries and epithelium of small airways of preterm lambs that were mechanically ventilated for 3 wk compared with control lambs born at term. No significant differences were detected for other proteins (inducible NOS, alpha-smooth muscle actin, and pancytokeratin). Lung vascular and respiratory tract resistances were greater in the chronically ventilated preterm lambs compared with control term lambs. These results support the notion that decreased eNOS in the pulmonary circulation and respiratory tract of preterm lambs may contribute to the pathophysiology of chronic lung disease.     

Phenylarsine oxide inhibits nitric oxide synthase in pulmonary artery endothelial cells

The role of protein tyrosine phosphorylation during regulation of NO synthase (eNOS) activity in endothelial cells is poorly understood. Studies to define this role have used inhibitors of tyrosine kinase or tyrosine phosphatase (TP). Phenylarsine oxide (PAO), an inhibitor of TP, has been reported to bind thiol groups, and recent work from our laboratory demonstrates that eNOS activity depends on thiol groups at its catalytic site. Therefore, we hypothesized that PAO may have a direct effect on eNOS activity. To test this, we measured (i) TP and eNOS activities both in total membrane fractions and in purified eNOS prepared from porcine pulmonary artery endothelial cells and (ii) sulfhydryl content and eNOS activity in purified bovine aortic eNOS expressed in Escherichia coli. High TP activity was detected in total membrane fractions, but no TP activity was detected in purified eNOS fractions. PAO caused a dose-dependent decrease in eNOS activity in total membrane and in purified eNOS fractions from porcine pulmonary artery endothelial cells, even though the latter had no detectable TP activity. PAO also caused a decrease in sulfhydryl content and eNOS activity in purified bovine eNOS. The reduction in eNOS sulfhydryl content and the inhibitory effect of PAO on eNOS activity were prevented by dithiothreitol, a disulfide-reducing agent. These results indicate that (i) PAO directly inhibits eNOS activity in endothelial cells by binding to thiol groups in the eNOS protein and (ii) results of studies using PAO to assess the role of protein tyrosine phosphorylation in regulating eNOS activity must be interpreted with great caution.     

Developmental changes in nitric oxide synthase isoform expression and nitric oxide production in fetal baboon lung

Nitric oxide (NO), produced by NO synthase (NOS), plays a critical role in multiple processes in the lung during the perinatal period. To better understand the regulation of pulmonary NO production in the developing primate, we determined the cell specificity and developmental changes in NOS isoform expression and action in the lungs of third-trimester fetal baboons. Immunohistochemistry in lungs obtained at 175 days (d) of gestation (term = 185 d) revealed that all three NOS isoforms, neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), are primarily expressed in proximal airway epithelium. In proximal lung, there was a marked increase in total NOS enzymatic activity from 125 to 140 d gestation due to elevations in nNOS and eNOS, whereas iNOS expression and activity were minimal. Total NOS activity was constant from 140 to 175 d gestation, and during the latter stage (160-175 d gestation), a dramatic fall in nNOS and eNOS was replaced by a rise in iNOS. Studies done within 1 h of delivery at 125 or 140 d gestation revealed that the principal increase in NOS during the third trimester is associated with an elevation in exhaled NO levels, a decline in expiratory resistance, and greater pulmonary compliance. Thus, there are developmental increases in pulmonary NOS expression and NO production during the early third trimester in the primate that may enhance airway and parenchymal function in the immediate postnatal period.