Impaired NO signaling in small pulmonary arteries of chronically hypoxic newborn piglets

We performed studies to determine whether chronic hypoxia impairs nitric oxide (NO) signaling in resistance level pulmonary arteries (PAs) of newborn piglets. Piglets were maintained in room air (control) or hypoxia (11% O(2)) for either 3 (shorter exposure) or 10 (longer exposure) days. Responses of PAs to a nonselective NO synthase (NOS) antagonist, N(omega)-nitro-L-arginine methylester (L-NAME), a NOS-2-selective antagonist, aminoguanidine, and 7-nitroindazole, a NOS-1-selective antagonist, were measured. Levels of NOS isoforms and of two proteins involved in NOS signaling, heat shock protein (HSP) 90 and caveolin-1, were assessed in PA homogenates. PAs from all groups constricted to L-NAME but not to aminoguanidine or 7-nitroindazole. The magnitude of constriction to L-NAME was similar for PAs from control and hypoxic piglets of the shorter exposure period but was diminished for PAs from hypoxic compared with control piglets of the longer exposure period. NOS-3, HSP90, and caveolin-1 levels were similar in hypoxic and control PAs. These findings indicate that NOS-3, but not-NOS 2 or NOS-1, is involved with basal NO production in PAs from both control and hypoxic piglets. After 10 days of hypoxia, NO function is impaired in PAs despite preserved levels of NOS-3, HSP90, and caveolin-1. The development of NOS-3 dysfunction in resistance level PAs may contribute to the progression of chronic hypoxia-induced pulmonary hypertension in newborn piglets.     

L-Arginine increases nitric oxide production in isolated lungs of chronically hypoxic newborn pigs.

Previously, our laboratory found that pulmonary hypertension developed and lung nitric oxide (NO) production was reduced when piglets were exposed to chronic hypoxia (Fike CD, Kaplowitz MR, Thomas CJ, and Nelin LD. Am J Physiol Lung Cell Mol Physiol 274: L517-L526, 1998). The purposes of this study were to determine whether L-arginine addition augments NO production and to evaluate whether L-arginine uptake is impaired in isolated lungs of chronically hypoxic newborn piglets. Studies were performed by using 1- to 3-day-old piglets raised in room air (control) or 10% O(2) (chronic hypoxia) for 10-12 days. Lung NO production was assessed in isolated lungs from both groups by measuring the perfusate accumulation of nitrites and nitrates (collectively termed NO(-)(x)) before and after addition of L-arginine (10(-2) M) to the perfusate. The rate of perfusate NO(-)(x) accumulation increased by 220% (from 0.8 +/- 0.4 to 2.5 +/- 0.5 nmol/min, P < 0.05) after L-arginine addition to chronic hypoxic lungs but remained unchanged (3.2 +/- 0. 8 before vs. 3.3 +/- 0.4 nmol/min after L-arginine) in control lungs. In the second series of studies, L-arginine uptake was evaluated by measuring the perfusate concentration of L-[(3)H]arginine at fixed time intervals. The perfusate concentration of L-[(3)H]arginine at each time point was less (P < 0.05) in control than in chronic hypoxic lungs. Thus L-arginine uptake was impaired and may underlie in part the reduction in lung NO production that occurs when piglets are exposed to 10-12 days of chronic hypoxia. Moreover, these findings in isolated lungs lead to the possibility that L-arginine supplementation might increase in vivo lung NO production in piglets with chronic hypoxia-induced pulmonary hypertension.     

Arachidonic acid metabolites and an early stage of pulmonary hypertension in chronically hypoxic newborn pigs

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.     

Thromboxane inhibition reduces an early stage of chronic hypoxia-induced pulmonary hypertension in piglets.

The pulmonary vasoconstrictor, thromboxane, may contribute to the development of pulmonary hypertension. Our objective was to determine whether a combined thromboxane synthase inhibitor-receptor antagonist, terbogrel, prevents pulmonary hypertension and the development of aberrant pulmonary arterial responses in newborn piglets exposed to 3 days of hypoxia. Piglets were maintained in room air (control) or 11% O(2) (hypoxic) for 3 days. Some hypoxic piglets received terbogrel (10 mg/kg po bid). Pulmonary arterial pressure, pulmonary wedge pressure, and cardiac output were measured in anesthetized animals. A cannulated artery technique was used to measure responses to acetylcholine. Pulmonary vascular resistance for terbogrel-treated hypoxic piglets was almost one-half the value of untreated hypoxic piglets but remained greater than values for control piglets. Dilation to acetylcholine in preconstricted pulmonary arteries was greater for terbogrel-treated hypoxic than for untreated hypoxic piglets, but it was less for pulmonary arteries from both groups of hypoxic piglets than for control piglets. Terbogrel may ameliorate pulmonary artery dysfunction and attenuate the development of chronic hypoxia-induced pulmonary hypertension in newborns.     

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.     

Contributions of nitric oxide synthase isozymes to exhaled nitric oxide and hypoxic pulmonary vasoconstriction in rabbit lungs

We investigated the source(s) for exhaled nitric oxide (NO) in isolated, perfused rabbits lungs by using isozyme-specific nitric oxide synthase (NOS) inhibitors and antibodies. Each inhibitor was studied under normoxia and hypoxia. Only nitro-L-arginine methyl ester (L-NAME, a nonselective NOS inhibitor) reduced exhaled NO and increased hypoxic pulmonary vasoconstriction (HPV), in contrast to 1400W, an inhibitor of inducible NOS (iNOS), and 7-nitroindazole, an inhibitor of neuronal NOS (nNOS). Acetylcholine-mediated stimulation of vascular endothelial NOS (eNOS) increased exhaled NO and could only be inhibited by L-NAME. Selective inhibition of airway and alveolar epithelial NO production by nebulized L-NAME decreased exhaled NO and increased hypoxic pulmonary artery pressure. Immunohistochemistry demonstrated extensive staining for eNOS in the epithelia, vasculature, and lymphatic tissue. There was no staining for iNOS but moderate staining for nNOS in the ciliated cells of the epithelia, lymphoid tissue, and cartilage cells. Our findings show virtually all exhaled NO in the rabbit lung is produced by eNOS, which is present throughout the airways, alveoli, and vessels. Both vascular and epithelial-derived NO modulate HPV.     

Role of airway nitric oxide on the regulation of pulmonary circulation by carbon dioxide.

The effects of hypercapnia (CO(2)) confined to either the alveolar space or the intravascular perfusate on exhaled nitric oxide (NO), perfusate NO metabolites (NOx), and pulmonary arterial pressure (Ppa) were examined during normoxia and progressive 20-min hypoxia in isolated blood- and buffer-perfused rabbit lungs. In blood-perfused lungs, when alveolar CO(2) concentration was increased from 0 to 12%, exhaled NO decreased, whereas Ppa increased. Increments of intravascular CO(2) levels increased Ppa without changes in exhaled NO. In buffer-perfused lungs, alveolar CO(2) increased Ppa with reductions in both exhaled NO from 93.8 to 61.7 (SE) nl/min (P < 0.01) and perfusate NOx from 4.8 to 1.8 nmol/min (P < 0.01). In contrast, intravascular CO(2) did not affect either exhaled NO or Ppa despite a tendency for perfusate NOx to decline. Progressive hypoxia elevated Ppa by 28% from baseline with a reduction in exhaled NO during normocapnia. Alveolar hypercapnia enhanced hypoxic Ppa response up to 50% with a further decline in exhaled NO. Hypercapnia did not alter the apparent K(m) for O(2), whereas it significantly decreased the V(max) from 66.7 to 55.6 nl/min. These results suggest that alveolar CO(2) inhibits epithelial NO synthase activity noncompetitively and that the suppressed NO production by hypercapnia augments hypoxic pulmonary vasoconstriction, resulting in improved ventilation-perfusion matching.     

Cyclooxygenase contracting factors and altered pulmonary vascular responses in chronically hypoxic newborn pigs.

Pulmonary hypertension and blunted pulmonary vascular responses to ACh develop when newborn pigs are exposed to chronic hypoxia for 3 days. To determine whether a cyclooxygenase (COX)-dependent contracting factor, such as thromboxane, is involved with altered pulmonary vascular responses to ACh, newborn piglets were raised in 11% O(2) (hypoxic) or room air (control) for 3 days. Small pulmonary arteries (100-400 microm diameter) were cannulated and pressurized, and their responses to ACh were measured before and after either the COX inhibitor indomethacin; a thromboxane synthesis inhibitor, dazoxiben or feregrelate; or the thromboxane-PGH(2)-receptor antagonist SQ-29548. In control arteries, indomethacin reversed ACh responses from dilation to constriction. In contrast, hypoxic arteries constricted to ACh before indomethacin and dilated to ACh after indomethacin. Furthermore, ACh constriction in hypoxic arteries was nearly abolished by either dazoxiben, feregrelate, or SQ-29548. These findings suggest that thromboxane is the COX-dependent contracting factor that underlies the constrictor response to ACh that develops in small pulmonary arteries of piglets exposed to 3 days of hypoxia. The early development of thromboxane-mediated constriction may contribute to the pathogenesis of chronic hypoxia-induced pulmonary hypertension in newborns.     

Altered endothelium-dependent relaxations in lambs with high pulmonary blood flow and pulmonary hypertension

Congenital heart disease associated with increased pulmonary blood flow produces pulmonary hypertension. To characterize vascular alterations in the nitric oxide (NO)-cGMP cascade induced by increased pulmonary blood flow and pulmonary hypertension, 10 fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt). When the lambs were 4-6 wk of age, we assessed responses of pulmonary arteries (PAs) and pulmonary veins (PVs) isolated from lungs of control and shunted lambs. PVs from control and shunted lambs relaxed similarly to exogenous NO (S-nitrosyl-acetyl-penicillamine), to NO produced endogenously (zaprinast and A-23187), and to cGMP (atrial natriuretic peptide). In contrast, relaxations to A-23187 and zaprinast were blunted in PAs isolated from shunted lambs relative to controls. Inhibitors of NO synthase (NOS) and soluble guanylate cyclase constricted control but not shunt PAs, indicating reduced basal NOS activity in shunt PAs. Pretreatment of shunt PAs with the substrates L-arginine and sepiapterin, a precursor for tetrahydrobiopterin synthesis, did not augment A-23187 relaxations. However, pretreatment with superoxide dismutase and catalase significantly enhanced A-23187 relaxations in shunt PAs. We conclude that increased pulmonary blood flow induces an impairment of endothelium-dependent relaxation that is selective to PAs. The impaired relaxation may be mediated in part by excess superoxide production.     

Chronic hypoxia alters nitric oxide-dependent pulmonary vascular responses in lungs of newborn pigs

Fike, Candice D., and Mark R. Kaplowitz. Chronichypoxia alters nitric oxide-dependent pulmonary vascular responses inlungs of newborn pigs. J. Appl.Physiol. 81(5): 2078-2087, 1996.Almost all ofthe studies evaluating the effect of chronic hypoxia on lung nitricoxide production have been performed in adult animals. Because resultsof studies in adult lungs should not be extrapolated to represent thenewborn lung, we performed studies to determine whether decreasednitric oxide production might be involved in the pathogenesis ofchronic hypoxia-induced pulmonary hypertension in newborns. We keptnewborn pigs in chambers filled with room air (control) or 11-12%O₂ for either 3-5 (short) or10-12 (long) days. Using isolated lungs, we measured pulmonary vascular responses to agents that either stimulate or inhibit thesynthesis of nitric oxide. To define the vascular sites of alteredproduction of nitric oxide, we applied the micropuncture technique andmeasured small venular pressures before and after treatment with anitric oxide synthesis inhibitor. Pulmonary vascular responses toacetylcholine were blunted in chronically hypoxic piglets of both theshort and long groups. The nitric oxide synthesis inhibitor had adifferent effect in the lungs of control piglets than in those ofchronically hypoxic piglets of the long but not of the short group. Forthe long group, the nitric oxide synthesis inhibitors causedconstriction of both arteries and veins in lungs of control but not ofchronically hypoxic piglets. These findings support the idea thatdecreased pulmonary vascular nitric oxide production occurs withchronic hypoxia in newborn pigs and might therefore contribute to thepathogenesis of pulmonary hypertension in newborns.

     

Voltage-gated K+ channels at an early stage of chronic hypoxia-induced pulmonary hypertension in newborn piglets

Our purpose was to determine whether smooth muscle cell membrane properties are altered in small pulmonary arteries (SPA) of piglets at an early stage of pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for 3 days. A microelectrode technique was used to measure smooth muscle cell membrane potential (E(m)) in cannulated, pressurized SPA (100- to 300-microm diameter). SPA responses to the voltage-gated K(+) (K(V)) channel antagonist 4-aminopyridine (4-AP) and the K(V)1 family channel antagonist correolide were measured. Other SPA were used to assess amounts of K(V)1.2, K(V)1.5, and K(V)2.1 (immunoblot technique). E(m) was more positive in SPA of chronically hypoxic piglets than in SPA of comparable-age control piglets. The magnitude of constriction elicited by either 4-AP or correolide was diminished in SPA from hypoxic piglets. Abundances of K(V)1.2 were reduced, whereas abundances of both K(V)1.5 and K(V)2.1 were unaltered, in SPA from hypoxic piglets. At least partly because of reduced amounts of K(V)1.2, smooth muscle cell membrane properties are altered such that E(m) is depolarized and K(V) channel family function is impaired in SPA of piglets at an early stage of chronic hypoxia-induced pulmonary hypertension.     

Chronic O2 exposure in the newborn rat results in decreased pulmonary arterial nitric oxide release and altered smooth muscle response to isoprostane.

Chronic oxygen exposure in the newborn rat results in lung isoprostane formation, which may contribute to the pulmonary hypertension evident in this animal model. The purpose of this study was to investigate the pulmonary arterial smooth muscle responses to 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2a)) in newborn rats exposed to 60% O2 for 14 days. Because, in the adult rat, 8-iso-PGF(2alpha) may have a relaxant effect, mediated by nitric oxide (NO), we also sought to evaluate the pulmonary arterial NO synthase (NOS) protein content and NO release in the newborn exposed to chronic hyperoxia. Compared with air-exposed control animals, 8-iso-PGF(2a) induced a significantly greater force (P < 0.01) and reduced (P < 0.01) relaxation of precontracted pulmonary arteries in the 60% O2-treated animals. These changes were reproduced in control pulmonary arteries by NOS blockade by using NG-nitro-L-arginine methyl ester. Pulmonary arterial endothelial NOS was unaltered, but the inducible NOS protein content was significantly decreased (P < 0.01) in the experimental group. Pulmonary (P < 0.05) and aortic (P < 0.01) tissue ex vivo NO accumulation was significantly reduced in the 60% O2-treated animals. We speculate that impaired pulmonary vascular tissue NO metabolism after chronic O2 exposure potentiates 8-iso-PGF(2alpha)-induced vasoconstriction in the newborn rat, thus contributing to pulmonary hypertension.     

Sodium-Coupled Neutral Amino Acid Transporter 1 (SNAT1) Modulates L-Citrulline Transport and Nitric Oxide (NO) Signaling in Piglet Pulmonary Arterial Endothelial Cells

Rationale

There is evidence that impairments in nitric oxide (NO) signaling contribute to chronic hypoxia-induced pulmonary hypertension. The L-arginine-NO precursor, L-citrulline, has been shown to ameliorate pulmonary hypertension. Sodium-coupled neutral amino acid transporters (SNATs) are involved in the transport of L-citrulline into pulmonary arterial endothelial cells (PAECs). The functional link between the SNATs, L-citrulline, and NO signaling has not yet been explored.

Objective

We tested the hypothesis that changes in SNAT1 expression and transport function regulate NO production by modulating eNOS coupling in newborn piglet PAECs.

Methods and Results

A silencing RNA (siRNA) technique was used to assess the contribution of SNAT1 to NO production and eNOS coupling (eNOS dimer-to-monomer ratios) in PAECs from newborn piglets cultured under normoxic and hypoxic conditions in the presence and absence of L-citrulline. SNAT1 siRNA reduced basal NO production in normoxic PAECs and prevented L-citrulline-induced elevations in NO production in both normoxic and hypoxic PAECs. SNAT1 siRNA reduced basal eNOS dimer-to-monomer ratios in normoxic PAECs and prevented L-citrulline-induced increases in eNOS dimer-to-monomer ratios in hypoxic PAECs.

Conclusions

SNAT1 mediated L-citrulline transport modulates eNOS coupling and thus regulates NO production in hypoxic PAECs from newborn piglets. Strategies that increase SNAT1-mediated transport and supply of L-citrulline may serve as novel therapeutic approaches to enhance NO production in patients with pulmonary vascular disease.     

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.     

Upregulation of vascular calcium channels in neonatal piglets with hypoxia-induced pulmonary hypertension

Inhibition of voltage-gated, L-type Ca(2+) (Ca(L)) channels by clinical calcium channel blockers provides symptomatic improvement to some pediatric patients with pulmonary arterial hypertension (PAH). The present study investigated whether abnormalities of vascular Ca(L) channels contribute to the pathogenesis of neonatal PAH using a newborn piglet model of hypoxia-induced PAH. Neonatal piglets exposed to chronic hypoxia (CH) developed PAH by 21 days, which was evident as a 2.1-fold increase in pulmonary vascular resistance in vivo compared with piglets raised in normoxia (N). Transpulmonary pressures (DeltaPtp) in the corresponding isolated perfused lungs were 20.5 +/- 2.1 mmHg (CH) and 11.6 +/- 0.8 mmHg (N). Nifedipine reduced the elevated DeltaPtp in isolated lungs of CH piglets by 6.4 +/- 1.3 mmHg but only reduced DeltaPtp in lungs of N piglets by 1.9 +/- 0.2 mmHg. Small pulmonary arteries from CH piglets also demonstrated accentuated Ca(2+)-dependent contraction, and Ca(2+) channel current was 3.94-fold higher in the resident vascular muscle cells. Finally, although the level of mRNA encoding the pore-forming alpha(1C)-subunit of the Ca(L) channel was similar between small pulmonary arteries from N and CH piglets, a profound and persistent upregulation of the vascular alpha(1C) protein was detected by 10 days in CH piglets at a time when pulmonary vascular resistance was only mildly elevated. Thus chronic hypoxia in the neonate is associated with the anomalous upregulation of Ca(L) channels in small pulmonary arteries in vivo and the resulting abnormal Ca(2+)-dependent resistance may contribute to the pathogenesis of PAH.     

Maturational changes in the regulation of pulmonary vascular tone by nitric oxide in neonatal rats

Nitric oxide (NO) is an important regulator of vasomotor tone in the pulmonary circulation. We tested the hypothesis that the role NO plays in regulating vascular tone changes during early postnatal development. Isolated, perfused lungs from 7- and 14-day-old Sprague-Dawley rats were studied. Baseline total pulmonary vascular resistance (PVR) was not different between age groups. The addition of KCl to the perfusate caused a concentration-dependent increase in PVR that did not differ between age groups. However, the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine augmented the K(+)-induced increase in PVR in both groups, and the effect was greater in lungs from 14-day-old rats vs. 7-day-old rats. Lung levels of total endothelial, inducible, and neuronal NOS proteins were not different between groups; however, the production rate of exhaled NO was greater in lungs from 14-day-old rats compared with those of 7-day-old rats. Vasodilation to 0.1 microM of the NO donor spermine NONOate was greater in 14-day lungs than in 7-day lungs, and lung levels of both soluble guanylyl cyclase and cGMP were greater at 14 days than at 7 days. Vasodilation to 100 microM of the cGMP analog 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphate was greater in 7-day lungs than in 14-day lungs. Our results demonstrate that the pulmonary vascular bed depends more on NO production to modulate vascular tone at 14 days than at 7 days of age. The observed differences in NO sensitivity may be due to maturational increases in soluble guanylyl cyclase protein levels.     

Contribution of oxygen radicals to altered NO-dependent pulmonary vasodilation in acute and chronic hypoxia

Chronic hypoxia (CH) increases pulmonary arterial endothelial nitric oxide (NO) synthase (NOS) expression and augments endothelium-derived nitric oxide (EDNO)-dependent vasodilation, whereas vasodilatory responses to exogenous NO are attenuated in CH rat lungs. We hypothesized that reactive oxygen species (ROS) inhibit NO-dependent pulmonary vasodilation following CH. To test this hypothesis, we examined responses to the EDNO-dependent vasodilator endothelin-1 (ET-1) and the NO donor S-nitroso-N-acetyl penicillamine (SNAP) in isolated lungs from control and CH rats in the presence or absence of ROS scavengers under normoxic or hypoxic ventilation. NOS was inhibited in lungs used for SNAP experiments to eliminate influences of endogenously produced NO. Additionally, dichlorofluorescein (DCF) fluorescence was measured as an index of ROS levels in isolated pressurized small pulmonary arteries from each group. We found that acute hypoxia increased DCF fluorescence and attenuated vasodilatory responses to ET-1 in lungs from control rats. The addition of ROS scavengers augmented ET-1-induced vasodilation in lungs from both groups during hypoxic ventilation. In contrast, upon NOS inhibition, DCF fluorescence was elevated and SNAP-induced vasodilation diminished in arteries from CH rats during normoxia, whereas acute hypoxia decreased DCF fluorescence, which correlated with augmented reactivity to SNAP in both groups. ROS scavengers enhanced SNAP-induced vasodilation in normoxia-ventilated lungs from CH rats similar to effects of hypoxic ventilation. We conclude that inhibition of NOS during normoxia leads to greater ROS generation in lungs from both control and CH rats. Furthermore, NOS inhibition reveals an effect of acute hypoxia to diminish ROS levels and augment NO-mediated pulmonary vasodilation.     

Cyclooxygenase-2 and an early stage of chronic hypoxia-induced pulmonary hypertension in newborn pigs.

Our objective was to determine whether cyclooxygenase (COX)-2-dependent metabolites contribute to the altered pulmonary vascular responses that manifest in piglets with chronic hypoxia-induced pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for 3 days. The effect of the COX-2 selective inhibitor NS-398 on responses to arachidonic acid or acetylcholine (ACh) was measured in endothelium-intact and denuded pulmonary arteries (100- to 400-microm diameter). Pulmonary arterial production of the stable metabolites of thromboxane and prostacyclin was assessed in the presence and absence of NS-398. Dilation to arachidonic acid was greater for intact control than for intact hypoxic arteries, was unchanged by NS-398 in intact arteries of either group, and was augmented by NS-398 in denuded hypoxic arteries. ACh responses, which were dilation in intact control arteries but constriction in intact and denuded hypoxic arteries, were diminished by NS-398 treatment of all arteries. NS-398 reduced prostacyclin production by control pulmonary arteries and reduced thromboxane production by hypoxic pulmonary arteries. COX-2-dependent contracting factors, such as thromboxane, contribute to aberrant pulmonary arterial responses in piglets exposed to 3 days of hypoxia.     

What leads to different mediators of alkalosis-induced vasodilation in isolated and in situ pulmonary vessels?

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.     

Role of 12-lipoxygenase in hypoxia-induced rat pulmonary artery smooth muscle cell proliferation

The 12-lipoxygenase (12-LO) pathway of arachidonic acid metabolism stimulates cell growth and metastasis of various cancer cells and the 12-LO metabolite, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], enhances proliferation of aortic smooth muscle cells (SMCs). However, pulmonary vascular effects of 12-LO have not been previously studied. We sought evidence for a role of 12-LO and 12(S)-HETE in the development of hypoxia-induced pulmonary hypertension. We found that 12-LO gene and protein expression is elevated in lung homogenates of rats exposed to chronic hypoxia. Immunohistochemical staining with a 12-LO antibody revealed intense staining in endothelial cells of large pulmonary arteries, SMCs (and possibly endothelial cells) of medium and small-size pulmonary arteries and in alveolar walls of hypoxic lungs. 12-LO protein expression was increased in hypoxic cultured rat pulmonary artery SMCs. 12(S)-HETE at concentrations as low as 10(-5) microM stimulated proliferation of pulmonary artery SMCs. 12(S)-HETE induced ERK 1/ERK 2 phosphorylation but had no effect on p38 kinase expression as assessed by Western blotting. 12(S)-HETE-stimulated SMC proliferation was blocked by the MEK inhibitor PD-98059, but not by the p38 MAPK inhibitor SB-202190. Hypoxia (3%)-stimulated pulmonary artery SMC proliferation was blocked by both U0126, a MEK inhibitor, and baicalein, an inhibitor of 12-LO. We conclude that 12-LO and its product, 12(S)-HETE, are important intermediates in hypoxia-induced pulmonary artery SMC proliferation and may participate in hypoxia-induced pulmonary hypertension.