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Canadian Institutes of Health Research Group in Lung Development, Lung Biology and Integrative Biology Programmes, Hospital for Sick Children Research Institute, and Departments of Pediatrics, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8
Submitted 15 September 2003 ; accepted in final form 21 November 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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pulmonary vascular resistance; nitric oxide; nitric oxide synthase; phosphatidylinositol 3-kinase
Most of the reported data on pulmonary arterial smooth muscle contraction and relaxation potential derive from the study of isolated vascular segments. This approach may have significant limitations in that there is evidence of airway epithelium-derived factor(s) that may impact on the contractile potential of the nearby arteries. Fernandez et al. (8) have shown in adult animals that a vasoactive epithelium-derived inhibitory factor reduces the phenylephrine-induced tone in endothelium-denuded rat aorta preparations mounted coaxially within epithelium-intact guinea pig tracheal tissue. Rat tracheal airway smooth muscle did not relax in response to this epithelium-derived inhibitory factor (7), suggesting that the tone-modulating effect was vascular smooth muscle specific.
Given its common developmental origin, we hypothesized that a functional interaction between the airway and pulmonary vascular system exists and may play an important role in regional control of pulmonary vascular resistance. The presence and release of this epithelium-derived inhibitory factor in lower airways and its effect on the adjacent pulmonary arterial smooth muscle have not been previously evaluated. The rat pulmonary artery may respond to this factor distinctly from the aorta. Thus the purpose of this study was to evaluate the pulmonary arterial muscle response to agonist stimulation when studied in the presence of the attached bronchi. We further hypothesized that the putative epithelium-derived inhibitory factor relaxes pulmonary arteries through a mechanism involving nitric oxide (NO).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Institutional review. All procedures involving animals were conducted according to criteria established by the Canadian Council for Animal Care. Approval for the study was obtained from the Animal Care Review Committee of the Hospital for Sick Children Research Institute.
Animal preparation. Four- to eight-day-old newborn Sprague-Dawley rats (Charles River, ON, Canada) were killed with an overdose of pentobarbital sodium (50 mg/kg), and their lungs were removed immediately after death.
Organ bath studies. Fourth-generation left lung intralobar pulmonary artery ring segments (average diameter of 100 µm and length of 2 mm) were dissected free and mounted in a wire myograph in isolation with either the bronchi attached or the detached bronchi floating in the bath. In some preparations, the pulmonary artery endothelium was removed by abrasion with a human hair. In pilot experiments (n = 6), we documented that acetylcholine-induced relaxation of prestimulated (U-46619) pulmonary arteries subjected to the lumen abrasion protocol was reduced to 5% or less, thus confirming effective endothelium removal.
Isometric changes were digitized and recorded online (Myodaq, Danish Myo Technology, Aarhus, Denmark). Tissues were bathed in 5 ml of Krebs-Henseleit buffer (in mM: 115 NaCl, 25 NaHCO3, 1.38 NaHPO4, 2.51 KCl, 2.46 MgSO4, 1.91 CaCl2, and 5.56 dextrose) bubbled with air-6% CO2 and maintained at 37°C. After 1 h of equilibration, the optimal resting tension of the tissue was determined by repeated stimulation with 128 mM KCl until maximum active tension was reached. All subsequent force measurements were obtained at optimal resting tension. The optimal resting tension was directly proportional to age, was not affected by the presence of the attached bronchi, and varied between 1 and 2 mN.
Pulmonary vascular muscle force generation was evaluated by stimulating with KCl (15-86 mM) and the thromboxane mimetic U-46619 (10-9-10-6 M). Similarly, the bronchial muscle was stimulated with U-46619 and acetylcholine (ACh; 10-9-10-6 M). Contractile responses were expressed as millinewtons per square millimeter and normalized to the tissue cross-sectional area as follows: (width x diameter) x 2.
NO synthase (NOS) inhibition was accomplished with the nonspecific inhibitor NG-monomethyl-L-arginine (L-NMMA; 10-5 M) and the neuronal (nNOS)- and inducible NOS (iNOS)-specific inhibitors 7-nitroindazole (7-NINA; 10-4 M) and 1400W (10-6 M), respectively, as reported by others (25). Wortmannin (10-7 M) and indomethacin (10-5 M) were used for phosphatidylinositol 3 (PI3)-kinase and cyclooxygenase inhibition, respectively. These inhibitors have been proven by others to block their respective pathways at the molar concentrations used (15, 18).
Bronchial epithelium removal. The bronchi epithelium was mechanically removed by gentle abrasion of its lumen with a human hair after dissection. Effectiveness of epithelium removal was evaluated by hematoxylin and eosin staining.
Ex vivo vascular NO accumulation. NO accumulation from pulmonary arteries (4th generation) without and with the attached bronchi was measured polarographically at 37°C by using a sealed water-jacketed cell [NOCHM, World Precision Instruments (WPI), Sarasota, FL] fitted with a NO electrode (ISO-NOP, WPI) and monitor (NOMKD, WPI). Before each experiment, the electrode was calibrated at the same temperature by using a standard nitrite solution (WPI) of known concentration, according to the manufacturer's instructions. The tissue was placed in Krebs-Ringer phosphate buffer (in mM: 130 NaCl, 5 KCl, 1.4 CaCl2, 1.3 MgSO4, 10 Na2HPO4, 5 glucose, pH 7.4) equilibrated in 21% O2 and maintained at 37°C. After a 5-min equilibration period, NO accumulation was quantitated from the slope of the polarograph output over a 5-min period and expressed as nanomoles per minute.
Data analysis. Data were evaluated by Student's t-test or two-way ANOVA. Statistical significance was accepted if P < 0.05. All statistical analysis was performed with the Number Cruncher Statistical System (Kaysville, UT). Data are presented as means ± SE.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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As shown in Fig. 2, force generation in pulmonary arteries or arteries with the adjacent airway (bronchus) attached was measured after stimulation with U-46619 or KCl. For both agonists, the force generated by the pulmonary artery was significantly reduced in the presence of the adjacent bronchus (P < 0.01). In contrast, the reverse was not true in that the dose-response curves after U-46619 stimulation for the bronchus and bronchus with artery attached were not statistically different (Fig. 3, top). Identical results were obtained by using ACh as an alternate agonist (Fig. 3, bottom). Pulmonary arterial endothelium removal in preparations with the bronchi attached significantly increased the pulmonary artery muscle force (P < 0.01 compared with similar preparation with endothelium intact), but the U-46619-induced force remained significantly lower than for the artery alone (Fig. 4).
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To determine whether the bronchus-derived relaxant factor was NO, the same experiments were performed in the presence of the nonspecific NOS inhibitor, L-NMMA and the nNOS (7-NINA)- and iNOS (1400W)-specific inhibitors (Fig. 5). Addition of L-NMMA or 7-NINA, but not 1400W, to the bath increased the bronchi with artery attached force generation to a level similar to the single artery preparation (Fig. 5).
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To exclude the possibility of the bronchial-derived relaxant factor being a prostanoid, studies were also conducted in the presence of a cyclooxygenase inhibitor. Addition of indomethacin further reduced the artery with bronchi attached muscle force generation, negating this possibility (Fig. 6).
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Abrasion of the bronchial epithelium led to a significant increase (P < 0.01) in the pulmonary arterial force generated in response to U-46619 and KCl stimulation (Fig. 7). Removal of the airway epithelium, albeit not completely, is illustrated by the representative histology of the bronchus with epithelium intact and after mechanical abrasion (Fig. 8). Suggesting that the bronchial-derived relaxation is induced by a diffusible factor is the evidence shown in Fig. 9, where the presence of the detached adjacent bronchus in the muscle bath significantly reduces the pulmonary artery force generation in response to U-46619 stimulation.
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Finally, to evaluate the possibility of this bronchial-derived factor acting via NOS stimulation, we measured NO release in a single artery and pulmonary artery with bronchus preparations in the presence and absence of wortmannin, a PI3-kinase inhibitor. PI3-kinase has been previously shown to phosphorylate endothelial NOS, thereby increasing NO production (4). Although wortmannin had no effect on the single artery, it significantly decreased (P < 0.01) the NO release in the pulmonary artery with attached bronchi tissue (Fig. 10), indicating that bronchi-dependent increased NO production is mediated via the PI3-kinase pathway.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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The existence of an airway epithelium-derived relaxant factor was postulated >10 yr ago (7-9, 22, 24). This factor was found to relax the preconstricted smooth muscle in a coaxial bioassay system involving a segment of trachea mounted around an aortic ring, often from a different animal species than the former (8). The rat tracheal airway smooth muscle did not relax in response to this epithelium-derived inhibitory factor (7), suggesting a vascular smooth muscle-specific tone-modulating effect. This, together with unsuccessful attempts to block its effect, initially led to the conclusion that a nonprostanoid epithelium-derived factor distinct from NO induces relaxation of the endothelium denuded aorta via a mechanism not involving the guanosine 3',5'-cyclic monophosphate (cGMP) pathway (8). Subsequently, others (14, 23) have documented a significant increase in the endothelium-denuded rat aorta cGMP levels when the aortas were exposed to tracheal epithelium and after methacholine stimulation. Such evidence is supportive of NO being involved in this process.
Although this factor was first recognized in the airway epithelium to have a vascular smooth muscle-specific relaxant potential, its effect on the pulmonary vasculature was never evaluated. Addressing its potential physiological relevance, Goldie et al. (12) speculated that this factor had a vasodilator effect on bronchial vessels, but this was never evaluated. To the best of our knowledge, this is the first study to confirm that this is a soluble factor secreted by intralobar bronchi, which has a significant tone-modulating effect on the adjacent pulmonary arteries. The nature of this airway epithelium-derived factor remains unknown, but our data implicate the release of NO via PI3-kinase stimulation that results in NOS phosphorylation as a final common pathway.
Specific sites for PI3-kinase phosphorylation have been identified for eNOS. Phosphorylation at the serine residue 1177 promotes a twofold increase in eNOS activity and markedly enhances NO production (4, 19). Such phosphorylation can be triggered by stimulation of the pathway by factors potentially derived from the airway, such as platelet-derived growth factor and epidermal growth factor (5, 6). The data from this study corroborate previous reports (8) indicating that airway epithelium-derived factor relaxing effect is endothelium independent. NO has been shown to be present in the exhaled gas of human newborns, and its level is altered by mechanical ventilation (2, 3, 26). Thus the possibility that bronchial epithelial cells express eNOS and are possibly susceptible to phosphorylation resulting in increased NO release must be considered, because in some animal species, such as the rabbit, all exhaled NO is produced by eNOS (25).
In this study, we were able to increase the pulmonary artery with attached bronchi force generation by utilizing the nonselective NOS (L-NMMA) and nNOS-selective (7-NINA) inhibitors. No significant change in force generation was seen in the presence of the specific iNOS inhibitor 1400W at a concentration found by others to be inhibitory (25). NOS suppression of the decrease in agonist-stimulated force and PI-3-kinase-dependent increase NO accumulation of pulmonary arteries with bronchi attached strongly suggest that the bronchi relaxant factor acts via NOS phosphorylation. The fact that L-NMMA effectively blocks nNOS in the lung (16, 25) suggests that the bronchial-epithelial-derived relaxing factor acts via nNOS in this preparation.
nNOS is constitutively produced by the pulmonary vascular and airway smooth muscle as well as bronchial epithelium (21) and is involved in the NO-mediated pulmonary vascular resistance in the fetus and newborn. nNOS can be phosphorylated by the PI3-kinase pathway (18). Although evaluation of the pulmonary artery force generation in the presence of the PI-3-kinase inhibitor would have been of interest, this was not feasible because wortmannin is known to suppress KCl-induced contraction of rat aorta via myosin light chain kinase inhibition (17), making it unsuitable for this purpose.
Of note is the wortmannin-induced decrease in pulmonary artery tissue basal NO accumulation in the presence of the attached bronchi but not in the isolated preparation, as shown in Fig. 10. To account for this apparent discrepancy, we speculate the following. The pulmonary arterial basal NO release is mostly dependent on the eNOS and nNOS activity. The 7-NINA-induced increase in pulmonary artery muscle force generation in the presence of the attached bronchi, as shown in this study, suggests involvement and likely predominant phosphorylation of nNOS in this preparation. The same may not be the case for the isolated pulmonary artery tissue where NOS phosphorylation, if present, may involve the eNOS isoform. Wortmanin has been shown by others to inhibit vascular endothelial growth factor (VEGF) (11) but not bradykinin-induced eNOS phosphorylation (13). Thus wortmannin, under the experimental conditions utilized in this study, likely partially inhibited pulmonary arterial nNOS phosphorylation in the preparation with the bronchus attached but not eNOS in the isolated tissue.
The bronchial force generation in response to the thromboxane analog or ACh was not altered when evaluated with the adjacent pulmonary artery attached. Aside from its contracting effect on the airway muscle via muscarinic-receptor stimulation, ACh is also a powerful stimulant of the pulmonary artery eNOS, thus increasing NO release (19). In the present study, we have not directly evaluated whether the relaxant factor acts on the airway smooth muscle; however, the work of others (7) suggests that its effect is vascular smooth muscle specific. Therefore, the observed relaxant effect of the attached bronchi on the pulmonary artery force generation is neither related to our experimental setup nor due to increased NO availability from the adjacent bronchi. Lastly, the decreased force generation in pulmonary arteries with the attached bronchi is not related to a tethering effect, because the presence of a nonattached bronchus has a similar relaxing effect on the artery.
These possibilities, however, imply that NO is produced in airway epithelial cells and diffuses across the vascular and airway wall to relax vascular smooth muscle. Because NO is relatively stable in vitro and highly diffusible, one would expect airway smooth muscle also to be relaxed by this mechanism. Yet, previous evidence utilizing the coaxial system (7) suggests that the epithelium-derived factor does not relax the tracheal smooth muscle, suggesting that possibly the increased NO release originates from pulmonary arterial smooth muscle. Figure 11 illustrates the proposed mechanism accounting for the bronchial epithelial effect on the pulmonary arterial smooth muscle tone.
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Our novel finding that the airway-derived relaxing factor acts on the newborn pulmonary arterial smooth muscle is of considerable potential physiological relevance to the transitional circulation during the perinatal period. Prenatally, the pulmonary vascular resistance is very high, allowing only a fraction of the venous return to the heart to perfuse the lungs. Birth is associated with a marked decrease in the resistance to blood flow in the lungs through a mechanism that involves NO release (10). The factors responsible for the maintenance of a low pulmonary vascular resistance postnatally and their regional changes to optimize ventilation perfusion in the lung are not completely understood. It is possible that an epithelium-derived relaxing factor, as found in our studies, may play a significant role in the process.
In late gestation, the three NOS isoforms are abundantly present in the pulmonary vascular and airway tissue (1, 21, 27). Yet, significant changes in the lung distribution and protein content of the NOS isoforms have been reported in the perinatal period (20). If the epithelium-derived relaxing factor acts via NOS, as our data indicate, it is possible that changes in the expression and/or activity of one or more isoforms contribute to the modulation of pulmonary vascular resistance during this period. Thus further investigation is required to evaluate the presence of this factor and its relaxant effect on the pulmonary arteries of fetuses and adult animals and the NOS isoform involved in its signal transduction pathway.
In summary, we have demonstrated for the first time that a bronchial epithelium-derived factor is capable of reducing the adjacent pulmonary vascular smooth muscle tone in the newborn rat via a mechanism involving NOS phosphorylation and NO release. This process may play an important role in the regulation of pulmonary vascular resistance under physiological conditions, especially during the perinatal period.
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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