Role of neuronal nitric oxide synthase in regulation of vascular and ductus arteriosus tone in the ovine fetus

Robyn L. Rairigh1,2, Laurent Storme1,2, Thomas A. Parker1,2, Timothy D. le Cras1,3, Neil Markham1,3, Malathi Jakkula1,3, and Steven H. Abman1,3

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Nitric oxide (NO) is produced by NO synthase (NOS) and contributes to the regulation of vascular tone in the perinatal lung. Although the neuronal or type I NOS (NOS I) isoform has been identified in the fetal lung, it is not known whether NO produced by the NOS I isoform plays a role in fetal pulmonary vasoregulation. To study the potential contribution of NOS I in the regulation of basal fetal pulmonary vascular resistance (PVR), we studied the hemodynamic effects of a selective NOS I antagonist, 7-nitroindazole (7-NINA), and a nonselective NOS antagonist, N-nitro-L-arginine (L-NNA), in chronically prepared fetal lambs (mean age 128 ± 3 days, term 147 days). Brief intrapulmonary infusions of 7-NINA (1 mg) increased basal PVR by 37% (P < 0.05). The maximum increase in PVR occurred within 20 min after infusion, and PVR remained elevated for up to 60 min. Treatment with 7-NINA also increased the pressure gradient between the pulmonary artery and aorta, suggesting constriction of the ductus arteriosus (DA). To test whether 7-NINA treatment selectively inhibits the NOS I isoform, we studied the effects of 7-NINA and L-NNA on acetylcholine-induced pulmonary vasodilation. The vasodilator response to acetylcholine remained intact after treatment with 7-NINA but was completely inhibited after L-NNA, suggesting minimal effects on endothelial or type III NOS after 7-NINA infusion. Western blot analysis detected NOS I protein in the fetal lung and great vessels including the DA. NOS I protein was detected in intact and endothelium-denuded vessels, suggesting that NOS I is present in the medial or adventitial layer. We conclude that 7-NINA, a selective NOS I antagonist, increases basal PVR, systemic arterial pressure, and DA tone in the late-gestation fetus and that NOS I protein is present in the fetal lung and great vessels. We speculate that NOS I may contribute to NO production in the regulation of basal vascular tone in the pulmonary and systemic circulations and the DA.

pulmonary hypertension; persistent pulmonary hypertension of the newborn; lung development


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NITRIC OXIDE (NO) is produced by NO synthase (NOS) during the conversion of L-arginine to L-citrulline and plays an important role in the regulation of vascular tone in the developing lung circulation. NO modulates basal pulmonary vascular tone in the late-gestation fetus (1, 2, 12, 14) and mediates pulmonary vasodilation to several physiological and pharmacological stimuli such as acetylcholine (ACh), shear stress, and oxygen (1, 2, 12, 29, 40). NO production also contributes to the decrease in pulmonary vascular resistance (PVR) at the time of birth (1-3, 12, 16, 25, 44). Inhibition of NOS with L-arginine analogs increases basal PVR, selectively blocks endothelium-dependent vasodilation, and attenuates the fall in PVR at birth (1, 2, 12).

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) (17). Although past studies have presumed that the NOS III isoform is the predominant source of NO production in the perinatal pulmonary circulation (18, 32, 50), the arginine analogs that were used to inhibit NOS activity in the physiological studies were not isoform selective (1, 2, 12, 14, 16, 25, 29). Our laboratory (34) demonstrated that the NOS II isoform is constitutively expressed in the late-gestation ovine fetal lung and that intrapulmonary infusions of selective NOS II antagonists increase PVR in the late-gestation fetus. Recently, Rairigh et al. (35) demonstrated that selective NOS II antagonists attenuate shear stress-induced pulmonary vasodilation during acute partial compression of the ductus arteriosus (DA), whereas nonselective blockade with N-nitro-L-arginine (L-NNA) completely blocked shear stress-induced pulmonary vasodilation. These findings suggest that NOS II contributes to NO production in the normal fetal lung under basal conditions (34) and, in part, to the release of NO during acute shear stress (35).

NOS I has also been identified in lung neuronal, epithelial, and vascular smooth muscle cells, and expression increases with advancing fetal age (9, 24, 26, 32, 39, 41, 50). However, whether NOS I activity also contributes to NO production and vasoregulation of the pulmonary circulation is unknown. Therefore, we performed a series of experiments to determine whether NOS I activity could also be a source of NO production in the fetal pulmonary circulation. We studied the effects of a selective NOS I antagonist, 7-nitroindazole (7-NINA), which has been previously shown to selectively inhibit NOS I in other experimental models (4-6, 20, 22, 28, 30, 31). First, we studied the hemodynamic effects of 7-NINA in chronically prepared fetal lambs. In addition, we performed Western blot analysis to determine the presence of NOS I in the ovine fetal lung and great vessels. In this study, we report that a selective NOS I antagonist, 7-NINA, increased PVR, systemic arterial pressure, and the pressure gradient across the DA. These findings suggest that NOS I contributes to NO production in the fetal circulation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Surgical preparation and physiological measurements. All procedures and protocols were reviewed and approved by the Animal Care and Use Committee at the University of Colorado (Denver) Health Sciences Center. Mixed-breed (Columbia-Rambouillet, Nebeker Ranch, Lancaster, CA) pregnant ewes between 125 and 128 days gestation (term 147 days) were fasted 24 h before surgery. Ewes were sedated with intravenous pentobarbital sodium (2-4 mg) and anesthetized with 1% tetracaine hydrochloride (3 mg) by lumbar puncture. Preoperative doses of intramuscular ampicillin (500 mg) and gentamicin (2 g) were administered. The ewes were kept sedated with pentobarbital sodium but breathed spontaneously throughout surgery. Under sterile conditions, the left forelimb of the fetal lamb was delivered through a uterine incision. A skin incision was made under the left forelimb after local infiltration with 1% lidocaine. 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 at the fifth to sixth intercostal space 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 with purse-string sutures. A 6-mm ultrasonic flow transducer (Transonics, Ithaca, NY) was placed around the LPA to measure blood flow to the left lung. The thoracotomy and skin incision were closed, and the fetus was gently placed in the uterus. 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 subcutaneously to an external pouch on the ewe. Ampicillin (250 mg) was infused daily into the fetal vein and amniotic cavity during the first 3 days after surgery. The animals were allowed at least 72 h for recovery from surgery before studies were performed. Catheter patency was ensured by daily infusions of heparinized saline (100 U/ml). The Ao, MPA, LA, and amniotic cavity catheters were connected to a computer-driven pressure transducer and recorder (Biopac Systems, Santa Barbara, CA). The 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) for continuous measurement of LPA flow. The absolute values of flow were determined from phasic blood flow signals as previously described (21, 27). PVR in the left lung was calculated with the following equation: PVR (in mmHg · ml-1 · min) = (mean MPA pressure - mean LA pressure)/LPA blood 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. 7-NINA (Sigma Pharmaceuticals, St. Louis, MO) at a dose of 1 mg was dissolved in 2 ml of sterile saline. L-NNA (Sigma Pharmaceuticals) at a dose of 20 mg was first dissolved in 1 M HCl, and then normal saline was added to achieve a final volume of 1 ml. Small amounts of NaOH (1 M) were used to slowly adjust the pH to 7.40. ACh (200 mg/ml; Sigma Pharmaceuticals) was dissolved in sterile saline and infused at a dose of 1.5 µg/min for 10 min before and after each of the study drugs (7-NINA and L-NNA).

Experimental design. The identical protocol was followed for each study drug: 7-NINA (1 mg; n = 4 animals, mean gestational age 128 ± 4 days) and L-NNA (20 mg; n = 4 animals, mean gestational age 133 ± 3 days). After 30 min of stable baseline hemodynamic measurements, ACh (1.5 µg/min for 10 min) was infused into the LPA with a precalibrated syringe infusion pump. Hemodynamic measurements were recorded at 10-min intervals for 30 min. 7-NINA and L-NNA was infused into the LPA over 10 min. Hemodynamic measurements were recorded at 10 min intervals for 30 min. A second dose of ACh (1.5 µg/min for 10 min) was infused into the LPA for comparison with the initial vasodilator response. Arterial blood gas tensions, pH, and oxygen saturation were measured from the Ao catheter at baseline and 30 min after infusion of each study drug.

Selection of the dose for each drug was based on published past studies (1, 2) and preliminary data. The dose of L-NNA used in this study has been shown to elevate PVR and inhibit ACh-induced pulmonary vasodilation for up to 3 h after treatment (1, 2). The ACh dose has been shown to consistently double the fetal pulmonary blood flow without changing pulmonary or Ao pressures (1, 2). The dose of 7-NINA was selected after preliminary dose-response studies showed that there were no hemodynamic effects of lower dosages (0.25 and 0.5 mg). The lowest dose of 7-NINA that altered the hemodynamics identified with a response in basal tone and a fully intact ACh-induced pulmonary vasodilation was 1 mg. Higher doses were avoided to minimize potential overlap with other NOS isoforms.

Western blot for NOS III and NOS I proteins. Western blot analysis was performed with 25 µg of fetal ovine lung, DA, MPA, and Ao and 25 µg of rat cerebellum protein according to a previously published method (25). NOS III was detected with a monoclonal antibody to NOS III (Transduction Laboratories, Lexington, KY). NOS I was detected with a polyclonal antibody to NOS I (Transduction Laboratories). The blots were blocked overnight with 5% nonfat dry milk at 4°C. NOS I antibody (1:3,000 dilution) in blocking solution was applied for 2 h. Horseradish peroxidase-conjugated secondary antibody (rabbit anti-goat IgG) was diluted to 1:15,000 in blocking solution and applied for 1 h. After being washed, the bands were visualized with enhanced chemiluminescence.

To determine whether NOS I protein was present in the endothelial or subendothelial layer, Western blot analysis for NOS I protein was performed on adjacent sections of the great vessels in which the endothelium was either intact or denuded. Sections of fetal ovine DA, MPA, and Ao were denuded by scraping with a scalpel to remove the endothelial layer. Western blot analysis for NOS I protein was performed with 25 µg of intact and denuded vessel sections.

Data analysis. Data are means ± SE. Statistical analysis was performed with the Statview 4.5 software package (Abacus Concepts, Berkeley, CA). Comparisons of two discrete time points were made with an analysis of variance for repeated measures with post hoc analysis. P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Hemodynamic effects of NOS I inhibition in the late-gestation fetus. 7-NINA increased mean pulmonary arterial pressure from 45 ± 3 (baseline) to 54 ± 2 (treatment) mmHg (P < 0.05) and caused a parallel rise in mean AoP from 43 ± 3 (baseline) to 48 ± 2 (treatment) mmHg (P < 0.01; Fig. 1). The pressure gradient between the pulmonary artery and Ao increased from 2 ± 1 to 7 ± 2 mmHg after the infusion of 7-NINA (P < 0.05). 7-NINA decreased LPA blood flow from 72 ± 8 (baseline) to 63 ± 6 (treatment) ml/min (P < 0.05; Fig. 1). At the doses used in this study, 7-NINA and L-NNA increased basal PVR by 37 and 72%, respectively (Fig. 2). The increase in PVR persisted up to 60 min after the infusion of 7-NINA. Arterial blood gas tensions, pH, oxygen saturation, mean LA pressure, and heart rate did not change after 7-NINA infusion (Table 1).


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Fig. 1.   Hemodynamic effects of 7-nitroindazole (7-NINA) in chronically prepared late-gestation ovine fetus. Brief infusions of 7-NINA (1 mg) increased mean pulmonary arterial pressure (PAP), aortic (Ao) pressure, and pressure gradient between pulmonary artery and Ao (A) and decreased left pulmonary arterial (LPA) blood flow (B).



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Fig. 2.   Effects of a selective nitric oxide synthase (NOS) I antagonist (7-NINA) and a nonselective NOS antagonist [N-nitro-L-arginine (L-NNA)] on fetal pulmonary vascular resistance (PVR). Brief infusion of each agent increased basal PVR.


                              
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Table 1.   Effects of 7-NINA and L-NNA on arterial blood gas tensions, pH, hemoglobin, O2 saturation, and LAP

Effects of selective NOS I and nonselective NOS inhibition on ACh-induced pulmonary vasodilation in the late-gestation fetus. During the control period before treatment with NOS antagonists, ACh decreased PVR by 41 ± 3 and 47 ± 11% before infusion of 7-NINA and L-NNA, respectively (P = not significant between groups; Fig. 3). After treatment with 7-NINA, the pulmonary vasodilator response to ACh remained intact. In contrast, the ACh-induced fall in PVR was completely blocked after treatment with the nonselective NOS antagonist L-NNA (Fig. 3). LPA blood flow increased from 72 ± 8 (baseline) to 132 ± 21 (treatment) ml/min (P < 0.05) after the initial infusion of ACh; after treatment with 7-NINA, a repeat dose of ACh increased LPA flow from 63 ± 6 (baseline) to 131 ± 18 (treatment) ml/min (P < 0.05; Table 2). In contrast, after treatment with L-NNA, a repeat dose of ACh did not increase LPA blood flow (50 ± 7 and 51 ± 5 ml/min for baseline and treatment, respectively; P = not significant; Table 2).


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Fig. 3.   Effects of a selective NOS I antagonist (7-NINA) and a nonselective NOS antagonist (L-NNA) on acetylcholine (ACh)-induced pulmonary vasodilation. Brief infusions of ACh decreased PVR by ~40% from baseline. After infusion of each study drug, ACh-induced pulmonary vasodilation was blocked after L-NNA but not by NOS I blocker. NS, not significant.


                              
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Table 2.   Effects of 7-NINA and L-NNA on ACh-induced pulmonary vasodilation on LPA blood flow, PAP, AoP, LAP, HR, pH, and arterial blood gas tensions

Western blot analysis of NOS I in the late-gestation fetus. NOS I protein in the nonsurgical fetal lamb was detected in the distal lung, Ao, MPA, and DA. NOS III protein was also detected (Fig. 4). NOS I protein was detected in both intact and denuded great vessels of the ovine fetus (Fig. 5), suggesting that NOS I is expressed in the medial and/or adventitial layer.


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Fig. 4.   Western blot of NOS I (neuronal; nNOS) and NOS III (endothelial; eNOS) protein content of late-gestation ovine fetal lung tissue and great vessels. NOS I polyclonal antibody detected a single band of 155 kDa in rat brain (Br; used as a positive control for NOS I) and in fetal lamb ductus arteriosus (DA), lung (Lu), main pulmonary artery (MPA), and Ao homogenates. NOS III monoclonal antibody detected a single band of 135 kDa.



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Fig. 5.   Western blot of NOS I protein content of intact and denuded ovine fetal great vessels. NOS I polyclonal antibody detected a single band of 155 kDa in intact and scraped fetal lamb Ao, MPA, and DA homogenates, indicating that NOS I is still present despite removal of endothelium.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We found that a selective NOS I inhibitor, 7-NINA, increased PVR and systemic arterial pressure in the chronically prepared fetal lamb. 7-NINA increased the pressure gradient between the pulmonary artery and Ao, suggesting an effect on DA tone. The nonselective NOS antagonist L-NNA also elevated PVR. However, in contrast to L-NNA, the NOS I antagonist caused less of an increase in basal PVR and maintained ACh-induced pulmonary vasodilation, suggesting that the effect of the NOS I inhibitor was not due to NOS III (endothelial) blockade. These findings suggest that the NOS I isoform contributes to basal production of NO and modulates pulmonary and systemic vascular tone as well as DA tone in the late-gestation fetus.

These findings are interesting because little is known about the role of NOS I in the regulation of vascular tone in the normal fetal lung. The physiological role of NOS I has been studied in other settings including experimental models of the modification of pain perception (19, 28, 30, 31), mediation of long-term potentiation of memory (10, 36), control of cerebral blood flow (20, 22, 33, 47, 48), and neurodegeneration after cerebral ischemia (13, 43, 45). NOS I has been localized to nonadrenergic noncholinergic nerves that can generate NO, which acts as a neurotransmitter to relax smooth muscle in the gastrointestinal and urogenital tracts and the airways (6-8, 11, 15, 36-39, 41, 46, 49). In the ovine fetal lung, NOS I is expressed in airway epithelium and vascular smooth muscle (39, 41). Previous studies have not studied the potential physiological role of NOS I in the fetal circulation. To test the hypothesis that NOS I activity regulates fetal hemodynamics, we infused a selective NOS I inhibitor directly into the LPA of the chronically prepared late-gestation ovine fetus. Our findings that a NOS I inhibitor (7-NINA) increased pulmonary arterial pressure and AoP and decreased LPA blood flow support the hypothesis that NOS I is active in the fetus. This inhibitor also increased the pressure gradient between the pulmonary artery and Ao, suggesting that NOS I inhibition caused mild changes in DA tone. However, further studies specifically addressing the role of NOS I on DA tone are needed. The increase in AoP after infusion of the NOS I antagonist also suggests a role for NOS I vasoregulation of the systemic circulation.

We also report that Western blot analysis demonstrated that NOS I protein is present in late-gestation fetal lung and great vessels. In addition, the NOS I protein was present in denuded Ao, MPA, and DA, suggesting that NOS I is expressed in the medial and/or adventitial layers. A study by Boulanger et al. (9) has shown that NOS I is expressed in cultured vascular smooth muscle cells, and recently, Sherman et al. (41) detected NOS I in vascular smooth muscle and airway epithelium of the fetal ovine lung. The physiological data presented in this study do not allow the specific identification of the cell type responsible for the NOS I activity. However, it is most likely that the hemodynamic changes seen in the pulmonary and systemic circulation as well as in the DA are due to the blockade of NOS I in the vascular smooth muscle.

Although each of the NOS inhibitors studied is competitive with L-arginine, their exact mechanism of action is unclear. The mechanism of inhibition of NOS activity by L-NNA (an L-arginine analog) involves binding the substrate site (42). 7-NINA (a nitrodazole) carries a net electronegative charge and is thought to bind to the heme prosthetic group of NOS, disrupting the flow of electrons through the enzyme and preventing NO formation (19, 23). 7-NINA also interferes with the binding of L-arginine by binding at the heme site, thereby competing with L-arginine (19, 23).

One of the potential limitations of pharmacological studies of NOS antagonists includes the relative selectivity for the NOS isoforms. Each agent has the potential at increased doses to inhibit NOS II and NOS III (4, 30, 31). In vitro studies (19) have shown that the concentrations of drug needed to achieve 50% inhibition (IC50) for 7-NINA is 0.047 µM. For comparison with L-arginine analogs (nonselective blockers that inhibit all NOS isoforms), 7-NINA was 1.8-fold more potent that N-nitro-L-arginine methyl ester (IC50 0.87 µM) and 5-fold more potent than N-monomethyl-L-arginine (IC50 2.37 µM) (30). Although high doses of 7-NINA have the potential to inhibit other NOS isoforms, in vitro studies have shown that 7-NINA does not inhibit ACh-induced endothelium-dependent vasodilation in isolated rabbit Ao preparations and does not affect mean arterial pressure in a range of species (5, 31, 51). In our study, we showed that 7-NINA elevated PVR without blocking ACh-induced pulmonary vasodilation in the late-gestation fetal lung, suggesting that 7-NINA inhibited NOS I selectively without affecting constitutive (endothelial) NOS III at the doses used in this study. We also showed that there was a significant increase in mean arterial pressure after the infusion of 7-NINA.

We conclude that a selective NOS I (neuronal) antagonist increases PVR and systemic arterial pressure without decreasing oxygenation in the late-gestation ovine fetus. NOS I inhibition also increased the pressure gradient between the pulmonary artery and Ao, suggesting that NOS I inhibition causes changes in DA tone. These findings support the hypothesis that NOS I or neuronal NOS contributes to the basal production of NO and modulation of basal vascular and DA tone in the normal fetal circulation. Whether stimulation of NOS I located in the vascular smooth muscle contributes to the release of NO and the marked decrease in PVR at birth is unknown.


    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 25 May 1999; accepted in final form 17 August 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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