Inducible NO synthase inhibition attenuates shear stress-induced pulmonary vasodilation in the ovine fetus

Robyn L. Rairigh1, Laurent Storme1, Thomas A. Parker1, Timothy D. le Cras2, John P. Kinsella1, Malathi Jakkula2, and Steven H. Abman3

2 Pediatric Heart Lung Center and Sections of 1 Neonatology and 3 Pulmonary and Critical Care Medicine, Department of Pediatrics, University of Colorado School of Medicine, Denver, Colorado 80218-1088


    ABSTRACT
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Recent studies have suggested that type II (inducible) nitric oxide (NO) synthase (NOS II) is present in the fetal lung, but its physiological roles are uncertain. Whether NOS II activity contributes to the NO-mediated fall in pulmonary vascular resistance (PVR) during shear stress-induced pulmonary vasodilation is unknown. We studied the hemodynamic effects of two selective NOS II antagonists [aminoguanidine (AG) and S-ethylisothiourea (EIT)], a nonselective NOS antagonist [nitro-L-arginine (L-NNA)], and a nonselective vasoconstrictor (U-46619) on PVR during partial compression of the ductus arteriosus (DA) in 20 chronically prepared fetal lambs (mean age 132 ± 2 days, term 147 days). At surgery, catheters were placed in the left pulmonary artery (LPA) for selective drug infusion, an ultrasonic flow transducer was placed on the LPA to measure blood flow, and an inflatable vascular occluder was placed loosely around the DA for compression. On alternate days, a brief intrapulmonary infusion of normal saline (control), AG, EIT, L-NNA, or U-46619 was infused in random order into the LPA. The DA was compressed to increase mean pulmonary arterial pressure (MPAP) 12-15 mmHg above baseline values and held constant for 30 min. In control studies, DA compression reduced PVR by 42% from baseline values (P < 0.01). L-NNA treatment completely blocked the fall in PVR during DA compression. AG and EIT attenuated the decrease in PVR by 30 and 19%, respectively (P < 0.05). Nonspecific elevation in PVR by U-46619 did not affect the fall in PVR during DA compression. Immunostaining for NOS II identified this isoform in airway epithelium and vascular smooth muscle in the late-gestation ovine fetal lung. We conclude that selective NOS II antagonists attenuate but do not block shear stress-induced vasodilation in the fetal lung. We speculate that stimulation of NOS II activity, perhaps from smooth muscle cells, contributes in part to the NO-mediated fall in PVR during shear stress-induced pulmonary vasodilation.

nitric oxide; pulmonary circulation; pulmonary hypertension; persistent pulmonary hypertension of the newborn; lung development


    INTRODUCTION
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

NITRIC OXIDE (NO) modulates basal pulmonary vascular tone in the late-gestation fetus (2, 10, 11) and mediates pulmonary vasodilation to several pharmacological and physiological stimuli such as acetylcholine (ACh), oxygen, and shear stress (2, 10, 26, 31, 34). At birth, the pulmonary circulation undergoes a dramatic fall in resistance that is, in part, due to stimulation of NO synthase (NOS) activity (2, 3, 10, 13, 23, 34). As shown by several studies (2, 10, 11, 13, 23, 26), inhibition of NOS with L-arginine analogs increases basal pulmonary vascular resistance (PVR), blocks the NO-mediated fall in PVR during shear stress-induced pulmonary vasodilation, and attenuates the fall in PVR at birth.

Three distinct isoforms of NOS have been identified: type I or neuronal (NOS I), type II or inducible (NOS II), and type III or endothelial (NOS III) (10). NOS I and NOS III are constitutive and regulated in part by calcium and calmodulin. Changes in expression of these NOS isoforms have been characterized during lung development in the rat and sheep fetus (3, 6, 17, 28, 37). NOS II is distinct from the other isoforms in that its activity is independent of intracellular calcium and it is regulated at the transcriptional level primarily through induction by endotoxin and cytokines (6). NOS II has been identified in the endothelium, epithelium, and macrophages in human and rat fetal lungs by immunostaining (22, 37). Although NOS II has been predominantly associated with pathophysiological conditions including septic shock, autoimmune disease, and inflammation (8, 12, 15, 19, 21, 25), its role in normal physiology is unknown.

Although past studies (17, 28, 37) have emphasized the potential role of the NOS III isoform as the source of vascular NO production in the perinatal lung, arginine analogs that were used to inhibit NOS activity in physiological studies were not isoform selective (2, 10, 11, 13, 23, 26). Whether other NOS isoforms, including NOS II, contribute to NO production in the normal perinatal pulmonary circulation is uncertain. A recent study (29) demonstrated that NOS II is present in fetal sheep lungs, and brief intrapulmonary infusions of selective NOS II antagonists increase basal PVR in the ovine fetus. These findings suggest that NOS II may also play a "constitutive" role and contribute to NO production in the normal fetal lung.

Although NOS II activity contributes to basal PVR, the effects of physiological studies on NOS II activity are unknown. A past study (10) demonstrated that inhibition of NOS blocks pulmonary vasodilation in response to increased shear stress. To test whether NOS II contributes in part to NO release during shear stress-induced pulmonary vasodilation, we studied the hemodynamic effects of two selective NOS II antagonists, aminoguanidine (AG) and S-ethylisothiourea (EIT) (5, 9, 14, 16, 20, 27, 35, 38), during partial compression of the ductus arteriosus (DA) in chronically prepared fetal lambs. We compared these responses with the effects of nitro-L-arginine (L-NNA), a nonselective NOS antagonist, and the effects of nonspecific elevation in basal PVR by U-46619 treatment. Furthermore, to determine the the localization of NOS II, we performed immunostaining on nonsurgical late-gestation ovine fetal lungs.


    METHODS
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Surgical preparation. All procedures and protocols were previously reviewed and approved by the Animal Care and Use Committee at the University of Colorado Health Sciences Center (Denver). Twenty mixed-breed (Columbia-Rambouillet, Nebekar Ranch) pregnant ewes between 125 and 128 days gestation (term 147 days) were fasted 24 h before surgery. The ewes were sedated with intravenous pentobarbital sodium (2-4 g) and anesthetized with 1% tetracaine hydrochloride (3 mg) by lumbar puncture. The ewes were kept sedated with pentobaribital sodium but breathed spontaneously throughout the surgery. Penicillin (500 mg) and gentamicin (2 g) were administered to the ewes at surgery. Under sterile conditions, the fetal lamb's left forelimb was delivered through a uterine incision. A skin incision was made under the left forelimb after local infiltration with lidocaine (2-3 ml, 1% solution). Polyvinyl catheters were inserted into the axillary artery and vein and advanced into the ascending aorta (Ao) and superior vena cava, respectively. A left axillary to sternal thoracotomy exposed the heart and great arteries. Polyvinyl catheters were inserted into the left pulmonary artery (LPA), main pulmonary artery (MPA), and left atrium (LA) by direct puncture and secured in position by purse-string catheters. The catheters were guided into position with a 14- or 16-gauge intravenous placement unit (Angiocath, Travenol, Deerfield, IL). The LPA catheter was inserted at the bifurcation of the MPA and the DA and guided through the common pulmonary artery into the LPA. The MPA catheter was inserted between the DA and the pulmonic valve. A 10-mm inflatable occluder (In Vivo Metric, Heldsburg, CA) was loosely placed around the DA to avoid any compression before the study period (2). A 6-mm ultrasonic flow transducer (Transonics Systems, Ithaca, NY) was placed around the LPA to measure blood flow to the left lung. The thoracotomy and skin incision were closed. The uteroplacental circulation was kept intact, and the fetus was gently placed in the uterus, with exposed surfaces bathed in warm towels. A catheter was placed in the amniotic space, ampicillin (500 mg) was administered into the amniotic cavity, and the hysterotomy was closed. The catheters and flow transducer cable were tunneled subcutaneouly to an external pouch on the ewe. Ampicillin (500 mg) was infused daily in the fetal vein and amniotic cavity during the first 3 days after surgery. The ewe was allowed to recover, and no further anesthesia was given. The animals were allowed at least 48-72 h for recovery from the surgery before studies were performed. Catheter patency was ensured by daily infusions of heparinized saline (100 U/ml).

Physiological measurements. The Ao, MPA, LA, and amniotic cavity catheters were connected to a computer-driven pressure transducer and recorder (Biopac Systems, Santa Barbara, CA). Pressures were referenced to the amniotic cavity pressure. The pressure transducer was calibrated with a mercury column manometer. The flow transducer cable was attached to an internally calibrated flowmeter (Transonics Systems) for continuous measurement of LPA flow. The absolute values of flow were determined from phasic blood flow signals as previously described (18, 24). PVR in the left lung was calculated by the following equation: PVR (in mmHg · ml-1 · min) = (mean MPA pressure - mean LA pressure)/LPA flow. Arterial blood gas tensions, pH, hemoglobin, and oxygen saturation were measured from blood samples that were drawn from the Ao catheter and measured at 39.5°C with a blood gas analyzer and hemoximeter (OSM-3, Radiometer, Copenhagen, Denmark).

Drug preparation. All drugs were freshly prepared on the day of study. Aminoguanidine hydrochloride (Tocris Cookson, St. Louis, MO) at a dose of 140 mg was dissolved in 2 ml of sterile saline. EIT (Tocris Cookson) at a dose of 0.12 mg was dissolved in 2 ml of sterile saline. L-NNA (Sigma Pharmaceuticals, St. Louis, MO) at a dose of 20 mg was dissolved with 1 M HCl, then 1 ml of normal saline was added. NaOH (1 M) was added to titrate the pH to 7.40. U-46619 (9,11-dideoxy-9alpha ,11alpha -epoxymethanoprostaglandin F2alpha ; Sigma Pharmaceuticals) at a dose of 0.01-0.25 µg/min (diluted in sterile saline) was titrated to increase basal PVR by 50%.

Experimental design. Studies were performed after at least 48 h of recovery from surgery. Study drugs were infused in random order on separate days, with a 1-day recovery between different drug studies. The identical protocol was followed for each drug: control (sterile saline; n = 4 animals, mean gestational age 130 days); L-NNA (20 mg; n = 4 animals, mean gestational age 130 days); AG (140 mg; n = 4 animals, mean gestational age 130 days); EIT (0.12 mg; n = 4 animals, mean gestational age 131 days); U-46619 (0.01-0.25 µg/min; n = 4 animals, mean gestational age 129 days). Hemodynamic measurements were recorded at 10-min intervals throughout the study period (baseline, drug infusion, ductal compression, and recovery). After 30 min of stable baseline hemodynamic measurements, one of the study drugs was infused into the LPA over 10 min. Immediately after the drug infusion was complete, the DA was compressed by inflation of the vascular occluder with saline, increasing mean pulmonary arterial pressure (MPAP) by 12-15 mmHg from baseline values, and held constant for 30 min. The selection of this level of MPAP was based on our previous experience in which pulmonary blood flow doubles by 30 min wthout causing changes in arterial blood gas tensions, pH, or aortic pressure (AoP) (1). The increased MPAP was kept constant during the study period by gently adjusting the degree of inflation as needed. Fluctuations in pressure were minimized by continuously monitoring MPAP. Heart rate, LPA blood flow, AoP, and LA and amniotic cavity pressures were similarly recorded. After 30 min of ductal compression, the occluder was rapidly deflated. Arterial blood gas tensions, pH, and oxygen saturation were measured at baseline, after 30 min of partial ductal compression, and after 30 min of recovery.

The dosage for each drug was based on previous studies and those published in the literature (9, 10, 29). The dose of L-NNA was based on a past study (2) that demonstrated a sustained pulmonary and systemic hypertension and a decrease in pulmonary blood flow with inhibition of ACh-induced pulmonary vasodilation. Selection of the doses for AG and EIT was based on a past study (29) demonstrating an increase in pulmonary and systemic pressures and a decrease in pulmonary blood flow without affecting ACh-induced pulmonary vasodilation, suggesting that AG and EIT selectively inhibit NOS II. U-46619 infusion was titrated to increase basal PVR by 50%.

Immunolocalization of NOS II protein in fetal lamb lung. To determine the sites of NOS II protein localization, we performed immunostaining of late-gestation fetal lung tissue that was obtained from nonsurgical fetal lambs (n = 4) at the same gestational age as the animals used for physiological studies. At autopsy, lungs were inflated by slow infusion of agarose (17). The pulmonary vasculature was perfused at 30-40 cmH2O pressure with saline, followed by buffered Formalin. Five-micrometer-thick paraffin sections were serially mounted onto Superfrost Plus slides (Fisher Scientific, Fairlawn, NJ). For NOS II immunostaining, the slides were dewaxed in 100% xylene and then rehydrated by immersion in 100% ethanol, 95% ethanol-5% water, 70% ethanol-30% water, and then 100% water. Antigen retrieval was performed by boiling the slides in 0.01 M citric acid, pH 6.0. The slides were washed in PBS (1× PBS is 2.7 mM KCl, 1.2 mM KH2PO4, 138 mM NaCl, and 8.1 mM Na2HPO4). Endogenous biotin in the tissue sections was blocked by glucose-glucose oxidase treatment [0.2 M glucose and 1.5 U/ml of glucose oxidase (Boehringer Mannheim Biochemicals) in 1× PBS]. The slides were washed in 1× PBS. The sections were blocked with Super Block (diluted 1:10 vol/vol in 1× PBS; Sky Tek, Logan, UT) and then incubated with an anti-NOS II primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:100 or an IgG1-negative control (Jackson Laboratories, West Grove, PA) in 1× PBS, 2% (wt/vol) bovine serum albumin (BSA), and 0.1% (wt/vol) NaN3. After incubation with the primary antibodies, the sections were washed in 1× PBS. Biotin-labeled anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA) was incubated with the sections at a dilution of 1:200 in 1× PBS, 2% (wt/vol) BSA, and 0.1% (wt/vol) NaN3. Again, the slides were washed in 1× PBS. The slides were incubated in streptavidin-biotin-horseradish peroxidase and developed with diaminobenzidine and hydrogen peroxide, with NiCl for enhancement (Vector). The NiCl enhancement-diaminobenzidine color development reaction was stopped by washing with water, and then the slides were dehydrated in 70% ethanol-30% water, 95% ethanol-5% water, 100% ethanol, and finally 100% xylene before coverslips were applied.

Data analysis. Data are presented as means ± SE. Statistical analysis was performed with the Statview 4.5 software package (Abacus Concepts, Berkeley, CA) and the analysis of variance SuperANOVA software package (Abacus Concepts). Comparisons were made by using univariate repeated-measures by linear-contrast analysis. P < 0.05 was considered significant.


    RESULTS
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Hemodynamic effects of partial compression of the DA. In control studies, infusions of normal saline (0.2 ml/min for 10 min) had no effects on MPAP, AoP, or LPA blood flow. After baseline measurements, the DA occluder was partially inflated to rapidly increase MPAP by 15 mmHg above baseline values. This pressure was kept constant for 30 min. In control (saline-treated) animals, LPA blood flow progressively increased above baseline by twofold during partial DA compression (P < 0.01). AoP did not change during the study period. Arterial blood gas tensions, pH, oxygen saturation, and heart rate did not change during or after partial compression of the DA (Table 1).

                              
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Table 1.   Effects of nonselective NOS blockade, selective NOS II blockade, and nonspecific elevation in PVR on arterial blood gas tensions, pH, hemoglobin, O2 saturation, and heart rate

Hemodynamic effects of a nonselective NOS antagonist on pulmonary vasodilation during partial compression of the DA. L-NNA infusion (0.2 ml/min for 10 min) into the LPA did not affect MPAP, AoP, LPA blood flow, or the pressure gradient between the pulmonary artery and the Ao. After infusions of L-NNA, the DA occluder was partially inflated to elevate MPAP to the same level achieved during the control study. In contrast with the twofold increase in LPA blood flow in control studies, L-NNA treatment inhibited the progressive pulmonary vasodilation after DA compression (Fig. 1). LPA blood flow at baseline was not different from measurements at 30 min of DA compression after L-NNA treatment [P = not significant (NS); Fig. 1]. At 30 min of DA compression, LPA blood flow in the L-NNA treatment group was nearly one-half of the blood flow in the control group (P < 0.001; Fig. 1). AoP, arterial blood gas tensions, pH, oxygen saturation, and heart rate did not change in this study group (Table 1).


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Fig. 1.   Effects of nonselective nitric oxide (NO) synthase (NOS) blockade on left pulmonary arterial (LPA) blood flow during 30 min of partial compression of ductus arteriosus (DA) in chronically prepared late-gestation fetus. Mean pulmonary arterial pressure (MPAP; top) and LPA blood flow (bottom) are shown during control and nitro-L-arginine (L-NNA) treatment periods. Brief infusions of L-NNA (20 mg) inhibited progressive increase in LPA blood flow, suggesting that NO mediates pulmonary vasodilation during partial compression of DA.

Hemodynamic effects of selective NOS II antagonists on pulmonary vasodilation during partial compression of the DA. AG infusion (0.2 ml/min for 10 min) into the LPA increased MPAP from 45 ± 2 to 52 ± 3 mmHg at 10 min (P < 0.05) and caused a parallel rise in mean AoP (41 ± 1 to 44 ± 1 mmHg; P < 0.05; Fig. 2). The pressure gradient between the pulmonary artery and the Ao did not change after the infusion of AG (Table 2). In comparison with control studies, AG attenuated the progressive pulmonary vasodilation after DA compression. After the infusion of AG, LPA blood flow increased from 76 ± 17 ml/min at baseline to 149 ± 25 ml/min after 30 min of DA compression (P < 0.01; Fig. 2). At 30 min of DA compression, LPA blood flow in the AG treatment group was 1.5-fold lower than blood flow in the control group (P < 0.01; Fig. 2). AoP, arterial blood gas tensions, pH, oxygen saturation, and heart rate did not change during partial compression of the DA (Table 1).


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Fig. 2.   Effects of selective NOS II blockade on LPA blood flow during 30 min of partial compression of DA in chronically prepared late-gestation fetus. MPAP (top) and LPA blood flow (bottom) are shown during control and aminoguanidine (AG) treatment periods. Brief infusions of AG (120 mg) increased MPAP before DA compression. AG treatment attenuated but did not block progressive increase in LPA blood flow, suggesting that NOS II contributes to NO-mediated pulmonary vasodilation during partial compression of DA.

                              
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Table 2.   Effects of nonselective NOS blockade, selective NOS II blockade, and nonspecific elevation in PVR on pressure gradient between pulmonary artery and aorta

EIT infusion (0.2 ml/min for 10 min) into the LPA increased MPAP from 47 ± 2 to 51 ± 2 mmHg (P < 0.05) before inflation of the DA occluder, and there was no change in mean AoP (P = NS; Fig. 3). The pressure gradient between the pulmonary artery and the Ao did not change significantly after infusion of EIT (Table 2). As observed with AG, EIT attenuated the progressive pulmonary vasodilation during DA compression. DA compression after EIT increased LPA blood flow from 80 ± 6 to 126 + 19 ml/min after 30 min (P < 0.01; Fig. 3). At 30 min of DA compression, LPA blood flow in the EIT treatment group was 1.6-fold lower than blood flow in the control group (P < 0.01; Fig. 3). AoP, arterial blood gas tensions, pH, oxygen saturation, and heart rate did not change during partial compression of the DA (Table 1).


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Fig. 3.   Effects of selective NOS II blockade on LPA blood flow during 30 min of partial compression of DA in chronically prepared late-gestation fetus. MPAP (top) and LPA blood flow (bottom) are shown during control and S-ethylisothiourea (EIT) treatment periods. Brief infusions of EIT (0.12 mg) increased MPAP before DA compression. EIT treatment attenuated but did not block progressive increase in LPA blood flow, suggesting that NOS II contributes to NO-mediated pulmonary vasodilation during partial compression of DA.

Hemodynamic effects of nonspecific elevation of basal PVR during partial compression of the DA. Continuous infusion of U-46619 (0.01-0.25 µg/min) increased MPAP from 49 ± 3 to 56 ± 1 mmHg after 10 min (P < 0.01) and caused a parallel rise in mean AoP from 45 ± 2 to 49 ± 3 mmHg (P < 0.01; Fig. 4). After a 50% increase in basal PVR was acheived, the DA occluder was partially inflated to elevate MPAP to the same level achieved during the control study. In contrast to the NOS antagonists, U-46619 did not affect progressive pulmonary vasodilation after DA compression. During DA compression, LPA blood flow increased from 117 ± 16 to 188 ± 9 ml/min (P < 0.01; Fig. 4). At 30 min of DA compression, LPA blood flow in the U-46619 treatment group was not significantly different from blood flow in the control group (P = NS; Fig. 4). AoP, arterial blood gas tensions, pH, oxygen saturation, and heart rate did not change during partial compression of the DA (Table 1).


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Fig. 4.   Effects of nonspecific elevation in basal pulmonary vascular resistance (PVR) on LPA blood flow during 30 min of partial compression of DA in chronically prepared late-gestation fetus. MPAP (top) and LPA blood flow (bottom) are shown during control and U-46619 treatment periods. Continuous infusion of U-46619 (0.01-0.25 µg/min) increased MPAP and decreased LPA blood flow to achieve an increase in basal PVR of 50% before DA compression. Nonspecific elevation of PVR by U-46619 did not affect progressive increase in LPA blood flow during partial compression of DA.

Comparisons of the effects on PVR during partial DA compression with the selective NOS II antagonists, the nonselective NOS antagonist, and the vasoconstrictor U-46619 are shown in Figs. 5-8. In control animals, PVR decreased during partial DA compression by 40% (P < 0.01). After the infusion of L-NNA, AG, EIT, and U-46619, basal PVR increased by 23, 32, 15, and 47%, respectively, before DA compression (P < 0.01). After 30 min of partial DA compression, PVR decreased by 40% in control animals (P < 0.01). AG and EIT attenuated the decrease in PVR (30 and 19%, respectively; P < 0.05) after 30 min of partial DA compression, whereas L-NNA completely blocked the decrease in PVR. Nonspecific vasoconstriction by U-46619 had no effect on pulmonary vasodilation during DA compression (Fig. 9).


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Fig. 5.   Effects of nonselective NOS blockade on PVR during partial compression of DA in chronically prepared late-gestation fetus. Brief infusions of L-NNA (20 mg) increased basal PVR by 23% before DA compression. L-NNA inhibited progressive fall in PVR during partial compression of DA.


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Fig. 6.   Effects of selective NOS II blockade on PVR during partial compression of DA in chronically prepared late-gestation fetus. Brief infusions of AG (120 mg) increased basal PVR by 32% before DA compression. AG attenuated but did not block progressive fall in PVR during partial compression of DA.


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Fig. 7.   Effects of selective NOS II blockade on PVR during partial compression of DA in chronically prepared late-gestation fetus. Brief infusions of EIT (0.12 mg) increased basal PVR by 15% before DA compression. EIT attenuated but did not block progressive fall in PVR during partial compression of DA.


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Fig. 8.   Effects of nonspecific elevation in basal PVR on PVR during partial compression of DA in chronically prepared late-gestation fetus. Continuous infusion of U-46619 (0.01-0.25 µg/min) increased basal PVR by 50% before DA compression. Nonspecific elevation of PVR did not affect progressive fall in PVR during partial compression of DA.


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Fig. 9.   Comparison of effects of nonselective NOS antagonist, selective NOS II antagonists, and nonspecific vasoconstriction during partial compression of DA. After 30 min of partial DA compression, AG and EIT attenuated decrease in PVR, whereas L-NNA completely blocked decrease in PVR. Nonspecific vasoconstriction by U-46619 had no effect on pulmonary vasodilation during partial compression of DA.

Immunolocalization of NOS II in the fetal lung is shown in Fig. 10. As illustrated, NOS II protein was detected in the airway epithelium and vascular smooth muscle. There was no staining of vascular endothelium or airway smooth muscle in any of the animals. Staining with an IgG control was negative.


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Fig. 10.   Immunolocalization of NOS II in late-gestation fetal lung tissue that was obtained from nonsurgical fetal lambs at same gestational age as animals used for physiological studies. A: low-power view illustrating distribution of NOS II in distal lung. Magnification, ×40. B: terminal bronchioli and accompanying small pulmonary artery. Magnification, ×100. C: high-power view of medium-size airway and accompanying artery. NOS II protein was detected in airway epithelium (solid arrow), with weaker staining of vascular smooth muscle (open arrow). Magnification, ×200. D: staining with an isotype-matched control antibody (IgG) was negative. Magnification, ×100.


    DISCUSSION
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

A past study (1) showed that acute compression of the DA progressively increases pulmonary blood flow and decreases PVR in the late-gestation fetus. The nonselective NOS antagonist L-NNA inhibits the rise in pulmonary blood flow and the decrease in PVR (10, 26), suggesting that an abrupt increase in flow or shear stress stimulated NO formation by one of the three known NOS isoforms. Although the endothelial or NOS III isoform is the likely source of NO in response to elevated shear stress flow, a recent study (29) demonstrated that NOS II mRNA is present in the ovine fetal lung and that selective NOS II antagonists increase basal PVR in the fetus. To determine whether physiological stimuli such as shear stress can stimulate NOS II activity, we studied the effects of selective NOS II antagonists on shear stress-induced fetal pulmonary vasodilation. We now report that two different selective NOS II antagonists (AG and EIT) attenuate the rise in pulmonary blood flow and decrease in PVR during partial DA compression. These findings suggest that shear stress stimulation of NO release is in part due to the activation of the NOS II isoform.

A previous study (4) demonstrated that maternal administration of selective NOS II antagonists caused contriction of the great vessels, including the DA, in the fetal rat. It is uncertain, however, whether these hemodynamic effects were due to direct blockade in the fetal lung or secondary to effects on the maternal or placental circulation, such as severe hypoxia or hypertension. A recent study (29) demonstrated that brief intrapulmonary infusions of three different selective NOS II antagonists did not increase the pressure gradient between the pulmonary artery and the Ao, suggesting a lack of significant effect on basal tone of the DA. We found that two selective NOS II antagonists (AG and EIT) did not increase the pressure gradient between the pulmonary artery and the Ao before partial compression of the DA.

We also report that NOS II protein is present in the late-gestation ovine fetal lung and predominantly localizes to airway epithelium and vascular smooth muscle. The strong immunostaining for NOS II was detected in the airway epithelium, with weaker staining of the vascular smooth muscle. The physiological data presented in this study do not allow the specific identification of the cell type responsible for the NOS II activity that caused NO release in response to shear stress. However, it is most likely that the hemodynamic changes caused by partial DA compression are more likely to be activation of NOS II in the vascular smooth muscle than in the airway epithelium. Shear stress predominantly acts on the vascular endothelium to stimulate NO release (35, 37). Whether shear stress or other physiological stimuli such as cyclic stretch or increased wall tension or pressure can act on vascular smooth muscle to release NO is uncertain.

These findings are interesting because little is known about the role of NOS II in the regulation of vascular tone in the normal fetal lung. Although past studies (17, 28, 37) have emphasized the role of the NOS III isoform as the source of vascular NO production in the perinatal lung, arginine analogs that were used to inhibit NOS activity in physiological studies were not isoform selective (2, 10, 11, 13, 23, 26). Whether other NOS isoforms including NOS II contribute to NO production in the normal perinatal pulmonary circulation is uncertain. Although NOS II has been shown to play a role in the pathophysiology of shock, autoimmune disease, and chronic inflammation (8, 12, 19, 21), few studies have expanded its potential physiological roles in normal circulation. A recent study (29) showed that NOS II may contribute to the regulation of basal vascular tone in the normal developing fetal lung. These data provide further support for the hypothesis that the NOS II ("inducible") isoform is constitutively expressed and that it actively produces NO under basal and stimulated conditions in the fetal pulmonary circulation.

Shear stress- or flow-induced vasodilation is mediated by the release of vasoactive substances such as NO, prostacyclin, and endothelium-dependent hyperpolarization factor from the endothelium (1, 7, 10, 30). Previous studies (1, 2, 10) that looked at shear stress-induced pulmonary vasodilation in the fetus were based on nonselective NOS blockade, thus inhibiting all three isoforms of NOS. With nonselective blockade of NOS with L-NNA, shear stress-induced pulmonary vasodilation is completely blocked. Two different selective NOS II antagonists attenuated shear stress-induced vasodilation, suggesting that NOS II activity may contribute to NO production during shear stress-induced vasodilation. Shear stress (3-24 h) has been shown to enhance expression of NOS III mRNA in a dose-dependent manner and increase the capacity of endothelial cells to release NO chronically (36). In cell culture studies of the acute endothelial cell response to the onset of flow, there is an abrupt increase in NO release (33), with an increase of several orders of magnitude in NO release over the shear stress range. This provides evidence of shear stress as a very sensitive regulator of NO. During acute partial compression of the DA, an abrupt increase in blood flow is more likely due to acute NO release than to transcriptional upregulation of NOS III. The increase in blood flow (shear stress) along the endothelial surface causes the release of NO from NOS III, which may contribute to pulmonary vasodilation (10). This same increase in blood flow may also stimulate NOS II in the vascular smooth muscle to release NO, contributing to NO-mediated pulmonary vasodilation in an autocrine pathway. Alternatively, diffusion of NO produced from airway epithelium could act on smooth muscle cells of adjacent pulmonary arteries, but this seems less plausible. We speculate, however, that perhaps ventilation-induced release of NO (10) could directly stimulate epithelial NOS II activity.

Each of the NOS II antagonists studied is competitive with L-arginine, but the exact mechanism of action remains unclear. AG, a non-amino acid inhibitor, is thought to inhibit NOS II by binding as a ligand to the heme iron at the catalytic site because AG deactivates other iron- or copper-containg enzymes in this manner (32). The isothiourea EIT has been shown to alter the heme spectra of NOS, suggesting binding to or interaction with the heme center (14, 32). Each agent has the potential at high doses to also inhibit NOS I and NOS III activities (27, 35). AG has been previously shown to have no effect on ACh-induced vasodilation, suggesting that AG inhibits NOS II selectively without blocking NOS III (9, 16, 20, 38). EIT has been described as a very potent and selective inhibitor of NOS II (5, 14, 27, 34). In vitro studies (27, 35) showed that AG and EIT are 80- and 30-fold more potent for NOS II than for NOS III, respectively. The IC50 for EIT was 13 nM, indicating that EIT is ~1,000-fold more potent than AG (IC50 value of 12 µM) (27, 35). A recent study (29) showed that AG and EIT elevated fetal PVR without blocking ACh-induced pulmonary vasodilation, suggesting that these inhibitors blocked NOS II selectively without affecting NOS III. The doses of AG and EIT used in our study were previously described to have no effect on ACh-induced pulmonary vasodilation, suggesting that they are selective for NOS II inhibition (29).

We conclude that nonselective blockade of NOS inhibits shear stress-induced pulmonary vasodilation in the late-gestation fetal lamb. Selective blockade of NOS II (inducible) attenuates shear stress-induced vasodilation. Nonspecific pharmacological elevation in basal PVR does not block shear stress-induced vasodilation. These findings support the hypothesis that NOS II activity contributes to the NO-mediated shear stress-induced pulmonary vasodilation. NOS II located in the smooth muscle cell and airway epithelium may contribute to the release of NO and the marked decrease in PVR at birth.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. L. Rairigh, Neonatology, Box B070, The Children's Hospital, 1056 E. Nineteenth Ave., Denver, CO 80218-1088.

Received 17 August 1998; accepted in final form 9 December 1998.


    REFERENCES
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

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