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Am J Physiol Lung Cell Mol Physiol 291: L473-L478, 2006. First published March 31, 2006; doi:10.1152/ajplung.00313.2005
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Adjacent bronchus attenuates pulmonary arterial contractility

Satyan Lakshminrusimha,1 James A. Russell,1,2 Sylvia F. Gugino,2 Rita M. Ryan,1 Bobby Mathew,1 Lori C. Nielsen,1 and Frederick C. Morin, III1

Departments of 1Pediatrics and 2Physiology and Biophysics, State University of New York at Buffalo, Center for Developmental Biology of the Lung, Buffalo, New York

Submitted 18 July 2005 ; accepted in final form 17 March 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bronchus-derived relaxing factor (BrDRF) decreases contractility of newborn rat pulmonary arteries (PA) and is dependent on nitric oxide (NO) synthesis. In vivo, this factor appears to gain access via the adventitial side of the PA. However, the adventitia has been reported to be a barrier to NO. We studied the effect of an adjacent bronchus on PA contractility to norepinephrine in nine juvenile lambs in the presence and absence of inhibitors of the NO pathway (LNA, ODQ, and Rp-8-Br-PET-cGMPS), cytochrome P-450 inhibitor (17-ODYA), perivascular nerve activity blocker (TTX), and superoxide scavenger (tiron), and following disruption of bronchial epithelium. We also evaluated whether BrDRF was effective on both the endothelial and/or adventitial side of PA. Fifth-generation PA rings with and without an attached bronchus were contracted in standard baths with norepinephrine. PA were dissected, cut open, and placed in a sided chamber in which adventitial and endothelial sides of the PA were exposed to unattached bronchus separately. Norepinephrine (10–8 to 10–5 M) contractions were expressed as a fraction of maximal KCl (118 mM) contractions. Norepinephrine contractions were significantly reduced by the presence of an attached bronchus, an effect reversed by pretreatment with LNA, ODQ, and Rp-8-Br-PET-cGMPS, and removal of bronchial epithelium. Unattached bronchus in the bath perfusing the adventitial side was effective in inhibiting the contractile response in PA. NO gas relaxed PA when administered on the endothelial side only. We speculate that BrDRF is a diffusible factor that crosses the adventitia and stimulates production of NO within the PA.

nitric oxide; pulmonary vascular resistance; norepinephrine

THE CONTRACTILE RESPONSE of isolated pulmonary arteries (PA) has been well studied in perinatal, juvenile, and adult animals with and without pulmonary hypertension (2, 9, 14, 23, 31, 33). The close proximity of these arteries to airways raises the possibility of interaction between the two. Belik et al. (3, 4) recently reported the presence of a bronchus-derived relaxing factor (BrDRF) that reduced PA force generation in newborn rats and that nitric oxide (NO) synthesis is necessary for BrDRF to reduce tone. Similar effects of airway epithelium influencing adjacent arterial tone have been reported by others (10, 11). These studies were done in a standard single tissue bath chamber where BrDRF has access to both the endothelial and adventitial sides of the isolated PA. However, in vivo, BrDRF is presented to only the adventitial side of the PA. Previous experiments in our laboratory have shown that pulmonary arterial adventitia is a barrier to NO in rabbits (29). If BrDRF is NO or mediates its effect through NO, how does this NO cross the adventitial barrier? Moreover, the bronchial effect on adjacent PA contractility has not been studied in larger mammals such as lambs, whose pulmonary developmental physiology and weight correspond more closely to human neonates.

We sought to determine whether, similar to rodents, BrDRF is produced in juvenile ovine bronchi and if it reduces pulmonary vascular tone. We hypothesized that this factor would be effective on both the adventitial and endothelial sides of the PA and stimulates NO production within the PA after permeating the adventitial layer.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
This study was approved by the State University of New York at Buffalo Laboratory Animal Care Committee. Juvenile lambs (6 wk old, n = 9) were anesthetized with pentothal sodium (250 mg iv) and killed by rapid exsanguination through a cardiac puncture. The heart and lungs were removed en bloc, and fifth-generation PA with and without adjacent bronchi were isolated. Care was taken to preserve the bronchial epithelium and arterial endothelium as described here.

Materials

The following pharmacological agents were used: DL-propranolol (10–6 M), norepinephrine hydrochloride (NE), NO synthase (NOS) antagonist N{omega}-nitro-L-arginine (LNA, 10–3 M), NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP), cytochrome P-450 inhibitor 17-oxydecanoic acid (17-ODYA, 10–5 M), nerve membrane voltage-gated sodium channel inhibitor tetradotoxin (TTX, 10–7 M), the Ca2+-activated K+ channel blocker tetraethylammonium (TEA; 10–6 M), and the superoxide scavenger tiron (10–5 M). All drugs were obtained from Sigma-Aldrich (St. Louis, MO). LNA was dissolved in warmed Krebs solution. SNAP was dissolved in a small quantity of DMSO and further diluted with distilled water. All other drugs were dissolved in distilled water. A specific inhibitor of soluble guanylyl cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ) (13, 24), was obtained from Tocris Cookson (St. Louis, MO). The specific cGMP-protein kinase (PKG) inhibitor Rp-beta-phenyl-1-N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothionate (Rp-8-Br-PET-cGMPS; referred to as PET in the rest of this article) was purchased from Biolog Life Science Institute through Ruth Langhorst International Marketing (La Jolla, CA). This agent is a competitive inhibitor of PKG. The Ki for PKG I{alpha}, Ibeta, and II isozymes is 35, 30, and 450 nM, respectively, and the Ki for protein kinase A is 11 µM (6). This drug has excellent membrane permeability and effectively inhibits both PKG isoforms (I{alpha} and Ibeta) found in vascular smooth muscle (19). PET was dissolved first in a small quantity of DMSO and then diluted in distilled water. DMSO, at the concentration used, did not alter pulmonary arterial tone in the tissue bath. Experiments were performed in a dark room since LNA is sensitive to light. NO gas (1,000 ppm) was obtained from Matheson Gas Products (Twinsburg, OH).

Standard Tissue Bath Studies

Technique. The heart and lungs were removed en bloc, and fifth-generation PA (inner diameters of ~0.5–1 mm) were dissected, isolated, and cut into rings as described previously (28). Some rings were carefully dissected with an attached bronchus. Rings were suspended in water-jacketed chambers filled with aerated (94% O2-6% CO2) modified Krebs-Ringer solution (in mM: 118 sodium chloride, 4.7 potassium chloride, 2.5 calcium chloride, 1.2 magnesium sulfate, 1.2 potassium biphosphate, 25.5 sodium bicarbonate, and 5.6 glucose). A continuous recording of isometric force generation was obtained by tying each vessel ring to a force displacement transducer (model UC2; Statham Instruments, Hato Rey, Puerto Rico) that was connected to a recorder (Gould Instrument Systems, Valley View, OH). After the arterial rings were mounted, they were allowed to equilibrate for 20 min in the bathing solution. A micrometer was used to stretch the tissues repeatedly in small increments over the following 45 min until resting tone remained stable at a passive tension of 0.8 g. Preliminary experiments determined that this procedure provided optimal length for generation of active tone to exogenous NE. Wet tissue weights, width, and length were obtained at the end of each experiment.

Protocols. Isolated PA and PA with attached bronchi were pretreated with propranolol (10–6 M) to block beta-adrenergic receptors. The arteries were contracted with increasing doses of NE (10–8 to 10–6 M). The arteries were then washed, and when their tone returned to baseline, they were constricted with 118 mM of potassium chloride (KCl). Contractions were recorded as milligram force and were normalized to milligrams of PA weight, cross-sectional area (length x width x 2 in mm2), and maximal response to 118 mM of KCl. Data in the figures are represented as a fraction of NE to maximal KCl contraction. Data normalized to weight and cross-sectional area (mg/mm2) showed similar changes in all studies and are not shown.

The bronchial epithelium was carefully denuded using a curved forceps in some PA with attached bronchi. Some PA and PA with attached bronchi were pretreated with LNA, ODQ, PET, tiron, TTX, TEA, and 17-ODYA and then contracted with NE. Some PA were contracted with an EC50 of NE (3 x 10–7 M) and relaxed with SNAP (10–8 to 10–5 M) after pretreatment with LNA, ODQ, and PET to study the effectiveness of these inhibitors in juvenile lambs. Relaxation responses are expressed as percent NE contraction.

Two-Sided Tissue Bath Studies

Technique. Fourth-generation pulmonary arterial segments with inside diameters of 2–3 mm were dissected into rings ~5 mm long. Each ring was then cut longitudinally and flattened out. In some pulmonary arterial segments, an attached bronchus was left in situ (PA+Attach Br).

Each cut pulmonary arterial segment was placed in a specially designed and constructed lucite tissue holder (Fig. 1) as described previously (29). This holder permits separate perfusion of the endothelial and adventitial surfaces while allowing direct measurement of the force of contraction. The tissue holder consists of upper and lower chambers that can be clamped together with the vessel sandwiched in between, thus exposing a round 0.125-cm2 area on opposite sides of the tissue. The upper and lower surfaces of the tissue are independently perfused at a rate of 5 ml/min with Krebs-Ringer solution with the use of a variable speed peristalitic pump (Minipuls 3; Gilson Medical Electronics, Middleton, WI) installed between the tissue holder and two glass reservoirs (1 each for upper and lower chambers). Total circulating volume in each chamber-reservoir system was 6 ml. An aerator in each reservoir was used to equilibrate the Krebs-Ringer solution with a gas mixture of 94% oxygen and 6% carbon dioxide. Each reservoir allowed free access for the addition of drugs or gases and fresh Krebs-Ringer solution as needed. The Krebs Ringer solution was maintained at 37°C by a heated water jacket around each reservoir as well as a heat exchanger placed in line just above the inlets to the upper and lower chambers. Four complete tissue holders were available permitting four pulmonary arterial segments from each lamb to be studied simultaneously.


Figure 1
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  		Fig. 1. Diagram of the tissue holder used for sided studies, to perfuse the endothelial and adventitial surfaces of juvenile ovine pulmonary arteries (PA). Modified Krebs solution entered the upper and lower chambers from the left side via a reservoir and heat exchanger in line with a recirculation roller pump. After passing over the upper or lower surfaces of the tissue, Krebs solution was returned to the reservoir for recirculation. A probe connected to a force transducer was used to depress the tissue and detect changes in the force of contraction. We studied 1) PA alone (with endothelial side facing downward and adventitial side facing upward); 2) PA with attached bronchus on the adventitial side (PA+Attach Br). A small aperture was made in the bronchial tissue to allow the force transducer to come in contact with the PA; 3) PA with unattached bronchus floating in the reservoir perfusing the lower chamber exposed to the endothelium [PA+Unattach Br (endo)]; and 4) PA with unattached bronchus floating in the reservoir perfusing the upper chamber exposed to the adventitia [PA+Unattach Br (adv)]. In experiments involving nitric oxide (NO) gas, NO was bubbled into the reservoir over a period of 15 s.

 
Protocols. To study pulmonary arterial contraction to NE and KCl, all pulmonary arterial segments were placed with the endothelial surface facing downward, as this was the only position in which we could mount an attached bronchus (Fig. 1). We studied 1) PA alone, 2) PA with an attached bronchus on the adventitial side, 3) PA with unattached bronchus floating in the reservoir perfusing the lower chamber exposed to the endothelium, and 4) PA with unattached bronchus floating in the reservoir perfusing the upper chamber exposed to adventitia. The pulmonary arterial segments were pretreated with propranolol (10–6 M) to block beta-adrenergic receptors. The arteries were first contracted with increasing concentrations of NE (10–8 to 10–5 M) added to the adventitial side. The arteries were then washed, unattached bronchial tissue was removed from the organ chambers, and when their tone returned to baseline, they were contracted with 118 mM of KCl. NE contractions were expressed as a fraction of KCl contractions. Results corrected to surface area and weight were similar and are not shown.

In the protocols evaluating relaxation responses to NO gas, the PA segments were half-maximally contracted with NE. These segments were placed either endothelial surface down or adventitial surface down, and NO gas was administered to the lower, closed chamber only (Fig. 1). NO gas (1,000 ppm) was diluted with nitrogen and administered through the aerator over a 15-s period in the following dilutions: 1 ml of 1:100, 1 ml of 1:10, 1 ml of 1:1, and last, 5 ml of 1:1 gas. Relaxation responses were expressed as % NE contraction.

Statistical Analysis

All data are expressed as means ± SE, with n representing the number of animals studied. Statistical comparisons of the curves were performed with one-way or repeated measures ANOVA as appropriate. Fisher's protected least significant differences post hoc test was used as needed to compare multiple groups. All statistical analyses were performed with StatView software (Abacus Concepts, Berkeley, CA). Significance was accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Standard Tissue Bath

The force generation in response to NE in PA with or without the adjacent bronchus attached is shown in Fig. 2. The presence of the adjacent bronchus significantly impaired the contraction to NE. The maximal contraction responses to KCl were not significantly different between the groups (192 ± 63 g/mm2 cross-sectional area in PA vs. 169 ± 35 g/mm2 in PA+Attach Br, P = 0.5).


Figure 2
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  		Fig. 2. Concentration-response curves of juvenile ovine PA and PA+Attach Br to norepinephrine (NE; 10–8 to 10–6 M) in a standard tissue bath. In some PA+Attach Br, bronchial epithelium was denuded [PA+Br (epi)(–)]. Data are represented as a fraction of NE contraction of the PA to maximal (Max) contraction obtained with 118 mM potassium chloride (KCl). All tissues were pretreated with propranolol (10–6 M). *P < 0.05 compared with PA + Attach Br by ANOVA repeated measures; n represents the number of lambs.

 
Removal of the bronchial epithelium significantly enhanced contraction of PA with an adjacent bronchus (Fig. 2). This contraction response was similar in magnitude to PA without an adjacent bronchus.

PA were contracted with EC50 of NE and relaxed with increasing concentrations of SNAP (Fig. 3). The NO pathway inhibitors ODQ and PET inhibited the relaxations to SNAP. LNA did not significantly alter the relaxations to SNAP. However, LNA completely blocked relaxations to acetylcholine and calcium ionophore A-23187 in these vessels (data not shown).


Figure 3
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  		Fig. 3. Relaxation responses to NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP; 10–8 to 10–5 M) in juvenile PA in the presence and absence of inhibitors of NO pathway. Pretreatment with N{omega}-nitro-L-arginine (LNA; 10–3 M) did not significantly alter the relaxation response to SNAP. However, pretreatment with soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ; 10–5 M) and protein kinase G inhibitor Rp-8-Br-PET-cGMPS (PET; 2 x 10–5 M) significantly inhibited relaxation responses to SNAP. *P < 0.05 compared with control curve. Relaxation data are represented as % of constriction with 3 x 10–7 M of NE.

 
Pretreatment of isolated PA with attached bronchi with the NOS inhibitor LNA significantly enhanced contraction to NE (Fig. 4). Their contraction becomes similar to PA without an attached bronchus. In contrast, pretreatment with LNA did not significantly enhance contractions to NE in PA without an attached bronchus.


Figure 4
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  		Fig. 4. Effect of pretreatment with NO synthase inhibitor LNA (10–3 M) on concentration-response curves to NE (10–8 to 10–6 M) from PA isolated from juvenile lambs. All tissues were pretreated with propranolol (10–6 M). *P < 0.05 compared with corresponding vessel without LNA by ANOVA repeated measures; n represents the number of lambs.

 
Similar to NOS inhibition, inhibition of sGC with ODQ enhanced contractions to NE in PA with attached bronchi (Fig. 5). However, significance was achieved only at the highest dose of NE. There was a trend toward increased contractions to NE in PA pretreated with ODQ, and statistical significance was reached at 3 x 10–7 M concentration of NE only.


Figure 5
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  		Fig. 5. Effect of pretreatment with sGC inhibitor ODQ (10–5 M) on concentration-response curves to NE (10–8 to 10–6 M) from PA isolated from juvenile lambs. Pretreatment with ODQ significantly improves the contraction response of PA with attached bronchus at 10–6 M of NE only. ODQ enhances contraction response in PA alone at 3 x 10–7 M of NE only. {dagger}P < 0.05 compared with corresponding vessel without ODQ by ANOVA with Fisher's protected least significant differences (PLSD) post hoc test; n represents the number of lambs.

 
PKG inhibition with PET enhanced contractions in PA with attached bronchi similar to those seen in PA alone (Fig. 6). Similar to pretreatment with LNA, PET did not significantly increase contractions in PA alone.


Figure 6
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  		Fig. 6. Effect of pretreatment with protein kinase G inhibitor PET (2 x 10–5 M) on concentration-response curves to NE (10–8 to 10–6 M) in juvenile PA. *P ≤ 0.05 compared with corresponding vessel without PET by ANOVA repeated measures; n represents the number of lambs.

 
Pretreatment of PA with attached bronchi with tiron, 17-ODYA, TEA, and TTX did not alter contraction response to NE (Fig. 7).


Figure 7
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  		Fig. 7. Effect of pretreatment with inhibitor of cytochrome P-450 pathway [17-oxydecanoic acid (17-ODYA)], superoxide scavenger (tiron), potassium channel blocker [tetraethylammonium (TEA)], and perivascular nerve activity blocker [tetradotoxin (TTX)] on concentration response to NE (10–6 M) in juvenile PA. *P ≤ 0.05 compared with PA+Br by ANOVA (n represents the number of lambs).

 
Two-Sided Tissue Bath

Pulmonary arterial contractile responses to adventitial addition of NE in the two-sided tissue bath are shown in Fig. 8. PA alone contracted well, reaching 83 ± 4% of the maximal KCl contraction with 10–5 M of NE. An attached bronchus reduced contractions to only 28 ± 12% of the maximal KCl contraction. An unattached bronchus in the bath perfusing the endothelial side inhibited contractions to NE at the highest concentration only. In contrast, an unattached bronchus in the bath perfusing the adventitial side inhibited contractions across the range of NE concentrations. The maximal contraction responses to KCl were not different between the groups.


Figure 8
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  		Fig. 8. Concentration-response curves to NE (10–8 to 10–5 M) in a sided chamber. Data are represented as a fraction of NE contraction of the PA to maximal contraction obtained with 118 mM potassium chloride. All tissues were pretreated with propranolol (10–6 M). *P < 0.05 compared with PA by ANOVA repeated measures. {dagger}P < 0.05 by ANOVA factorial with Fisher's PLSD post hoc test at that concentration only (n = 6 lambs for all groups).

 
Figure 9 shows the relaxation response of PA to NO gas in the two-sided chamber. Increasing concentrations of NO on the adventitial side did not relax PA. In contrast, NO gas administered to the endothelial side relaxed the PA.


Figure 9
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  		Fig. 9. Comparison of NO concentration-response curves between adventitial (Adv) and endothelial (Endo) exposure in juvenile ovine PA. The vessels were pretreated with propranolol and contracted with NE. Relaxations are expressed as percent NE contraction. *P < 0.01 by ANOVA repeated measures compared with adventitial addition; n represents the number of lambs.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown that NE-mediated pulmonary arterial contractions are reduced by an adjacent bronchus in juvenile lambs. In a series of elegant experiments, Belik et al. (4) had shown that this factor is produced by the bronchial epithelium, is independent of pulmonary arterial endothelium, and is mediated by NO through a mechanism involving NOS phosphorylation via phosphatidylinositol 3-kinase stimulation. The results from our study confirm the presence of this factor in a large mammalian species with close physiological resemblance to humans. We have also demonstrated that the effect of this factor is dependent on the NO-sGC-PKG-cGMP pathway. For the first time, we have shown that this factor, unlike NO, is effective on both the adventitial side and endothelial side of the PA.

We chose to normalize contraction to NE as a fraction of the maximal contraction to KCl. We also analyzed contraction data normalized to PA weight (in the case of PA with an attached bronchus, the bronchus was removed and the pulmonary arterial segment alone was weighed) and also to pulmonary arterial surface area. Data were similar with all forms of normalization; in fact, higher levels of significance were obtained with normalization to weight and surface area. KCl contractions, when expressed as mg/mg pulmonary arterial weight or mg/mm2 surface area, tended to be lower in the PA with attached bronchus compared with PA alone. So, by obtaining a ratio of NE contraction to maximal KCl contraction, we compared each tissue to its own maximal contractile capacity, thereby eliminating any bias generated by differences in weight, surface area, and mechanical interference by the attached bronchus.

The presence of an attached bronchus significantly inhibited contractions to NE (Fig. 2), an effect that has been observed by others (10, 26, 27). Removal of bronchial epithelium reverses this inhibition confirming it to be the source of this factor. The exact mechanism of action of the bronchial factor is controversial. Whereas some authors have suggested that this factor is a non-prostanoid factor distinct from NO and independent of cGMP (10), others have documented increased cGMP following stimulation with this factor (15), possibly by activation of particulate guanylyl cyclase and not NO-dependent sGC (15, 27). More recently, Belik et al. (4) demonstrated that pretreatment with the nonspecific NOS inhibitor NG-monomethyl-L-arginine and the neuronal NOS inhibitor 7-nitroindazole increased the force generated by PA with an attached bronchus to values comparable to PA alone. However, these authors did not provide data regarding the effect of such pretreatments on contractions of PA alone. We compared the effect of pretreatment with inhibitors of NOS, sGC, and PKG on both single PA and PA with attached bronchi. The effectiveness of ODQ and PET in blocking relaxations to a NO donor (SNAP) in juvenile ovine PA was demonstrated (Fig. 3). Similar results have been reported with the use of these agents in neonatal pulmonary vessels (12, 34). Inhibition of the three enzymes involved in NO-mediated relaxation resulted in a significant increase in NE-induced contractile force in PA with attached bronchi but not in single PA, suggesting that NO pathway is more active in the presence of an adjacent bronchus (Figs. 4–6).

A small difference in the contractile response to NE between PA with and without an adjacent bronchus persists after pretreatment with ODQ and PET (Figs. 5 and 6). We entertained the possibility that a non-NO factor such as endothelium- derived hyperpolarizing factor (EDHF) or a factor released by perivascular nerves mediated these effects in addition to NO. Possible candidates for EDHF include C-type natriuretic peptide (CNP) (7, 8), hydrogen peroxide (20, 21, 25), and products of cytochrome P-450 pathway (1). The effects of CNP are mediated through PKG and hence are inhibited by PET (18) and cannot explain the residual contraction following pretreatment with PET. Inhibitors of cytochrome P-450 pathway (17-ODYA), superoxide scavenger (tiron, which indirectly depletes endogenous hydrogen peroxide), and potassium channel blocker (TEA) and perivascular nerve activity blocker (TTX) did not increase contractions to NE in PA with attached bronchi (Fig. 7). An alternative explanation for the difference between PA alone and PA with attached bronchus pretreated with ODQ/PET could be that ODQ and PET (at the concentrations used in this study) only partly block relaxation response to NO (as shown in Fig. 3).

Experiments done in our laboratory and others have shown that the adventitia is a barrier to NO in rabbit PA (29) and rat aorta (35). We demonstrated a similar differential response to NO gas in juvenile ovine PA (Fig. 9). Administration of NO gas on the adventitial side did not produce any significant relaxation. In contrast, NO gas was an effective vasodilator when administered on the endothelial side of the PA. High levels of superoxide anions generated by NADPH oxidase in the adventitia constitute a barrier capable of inactivating NO (22). If BrDRF-mediated relaxation is via production of NO, can an adjacent bronchus by virtue of its anatomical location on the adventitial side of the PA exert its inhibitory effect in vivo? We answered this question using a double-sided chamber (Fig. 1). Unattached bronchus on the adventitial side inhibited contraction to NE in the PA similar to an attached bronchus (Fig. 8). The inhibition of contraction by an unattached bronchus on the adventitial side was at least as good as, if not better than, the inhibition of contraction by an unattached bronchus on the endothelial side. The inhibition observed with the unattached bronchus on the adventitial side coupled with the lack of relaxation to NO on that side appears to rule out NO as the BrDRF. This indicates that the BrDRF can effectively permeate thorough pulmonary arterial adventitia and activate NOS, possibly within the pulmonary arterial wall. Pulmonary arterial adventitia is a source of various enzymes and has been recently shown to be capable of increasing cGMP in the medial layer of rat aorta (5, 16, 17). Thus the adventitia could be a potential site of NOS activation by BrDRF.

There are several limitations to this study. Many laboratories, including ours, study isolated vessels in conventional tissue baths using a 94% oxygen and 6% CO2 gas mixture (23, 28, 32). The PO2 of the buffer solution bathing the tissue is close to 500 mmHg and the PCO2 is ~40 mmHg. This approach is based on the assumption that the inner core of smooth muscle cells in the vessel may be relatively hypoxic because of lack of perfusion through the vasa vasorum in the isolated vessel bath. It has been recently shown that bronchial inhibition of pulmonary arterial contraction is not observed when the bath solution is bubbled with nitrogen (3). We acknowledge that the oxygen environment of our vessels may have influenced some of our results. Second, the presence of an attached bronchus can mechanically interfere with the contraction response of the PA. We consider this possibility unlikely because of the limited area (usually <20% of the circumference of pulmonary arterial ring) of attachment. However, to eliminate any such bias, we used maximal KCl contraction generated by the PA and bronchus together to normalize the contraction responses to NE.

In summary, we have demonstrated that a BrDRF is active in juvenile ovine pulmonary circulation and is capable of reducing vascular smooth muscle tone in a NO-sGC-cGMP-mediated protein kinase-mediated mechanism. We also demonstrate that, in contrast to NO, this factor is diffusible across the adventitia. Thus as Belik et al. (4) hypothesize, BrDRF is not NO, but it probably stimulates NOS in the PA. This factor may play an important role in the regulation of pulmonary vascular tone.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Lakshminrusimha, Division of Neonatology, Women and Children's Hospital of Buffalo, 219 Bryant St., Buffalo, NY 14222 (E-mail: slakshmi{at}buffalo.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bauersachs J, Popp R, Fleming I, and Busse R. Nitric oxide and endothelium-derived hyperpolarizing factor: formation and interactions. Prostaglandins Leukot Essent Fatty Acids 57: 439–446, 1997.[CrossRef][Web of Science][Medline]
  2. Belik J. Large pulmonary arteries and the control of pulmonary vascular resistance in the newborn. Can J Physiol Pharmacol 72: 1464–1468, 1994.[Web of Science][Medline]
  3. Belik J, Pan J, Jankov RP, and Tanswell AK. Bronchial epithelium-associated pulmonary arterial muscle relaxation in the rat is absent in the fetus and suppressed by postnatal hypoxia. Am J Physiol Lung Cell Mol Physiol 288: L384–L389, 2005.[Abstract/Free Full Text]
  4. Belik J, Pan J, Jankov RP, and Tanswell AK. A bronchial epithelium-derived factor reduces pulmonary vascular tone in the newborn rat. J Appl Physiol 96: 1399–1405, 2004.[Abstract/Free Full Text]
  5. Beranova P, Schott C, Chalupsky K, Kleschyov AL, Stoclet JC, and Muller B. Role of the adventitia in the cyclic GMP-mediated relaxant effect of N-hydroxy-L-arginine in rat aorta. J Vasc Res 42: 331–336, 2005.[CrossRef][Web of Science][Medline]
  6. Butt E, Pohler D, Genieser HG, Huggins JP, and Bucher B. Inhibition of cyclic GMP-dependent protein kinase mediated effects by (Rp)-8-bromo-PET-cyclic GMPS. Br J Pharmacol 116: 3110–3116, 1995.[Web of Science][Medline]
  7. Chauhan SD, Hobbs AJ, and Ahluwalia A. C-type natriuretic peptide: new candidate for endothelium-derived hyperpolarising factor. Int J Biochem Cell Biol 36: 1878–1881, 2004.[CrossRef][Web of Science][Medline]
  8. Chauhan SD, Nilsson H, Ahluwalia A, and Hobbs AJ. Release of C-type natriuretic peptide accounts for the biological activity of endothelium-derived hyperpolarizing factor. Proc Natl Acad Sci USA 100: 1426–1431, 2003.[Abstract/Free Full Text]
  9. Dunn JA, Lorch V, and Sinha SN. Responses of small intrapulmonary arteries to vasoactive compounds in the fetal and neonatal lamb: norepinephrine, epinephrine, serotonin, and potassium chloride. Pediatr Res 25: 360–363, 1989.[Web of Science][Medline]
  10. Fernandes LB, Paterson JW, and Goldie RG. Co-axial bioassay of a smooth muscle relaxant factor released from guinea-pig tracheal epithelium. Br J Pharmacol 96: 117–124, 1989.[Web of Science][Medline]
  11. Fernandes LB, Preuss JM, Paterson JW, and Goldie RG. Epithelium-derived inhibitory factor in human bronchus. Eur J Pharmacol 187: 331–336, 1990.[CrossRef][Web of Science][Medline]
  12. Gao Y, Dhanakoti S, Tolsa JF, and Raj JU. Role of protein kinase G in nitric oxide- and cGMP-induced relaxation of newborn ovine pulmonary veins. J Appl Physiol 87: 993–998, 1999.[Abstract/Free Full Text]
  13. Garthwaite J, Southam E, Boulton CL, Nielsen EB, Schmidt K, and Mayer B. Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Mol Pharmacol 48: 184–188, 1995.[Abstract]
  14. Gonzalez-Luis G, Perez-Vizcaino F, Garcia-Munoz F, de Mey JG, Blanco CE, and Villamor E. Age-related differences in vasoconstrictor responses to isoprostanes in piglet pulmonary and mesenteric vascular smooth muscle. Pediatr Res 57: 845–852, 2005.[CrossRef][Web of Science][Medline]
  15. Hay DW, Muccitelli RM, Page CP, and Spina D. Correlation between airway epithelium-induced relaxation of rat aorta in the co-axial bioassay and cyclic nucleotide levels. Br J Pharmacol 105: 954–958, 1992.[Web of Science][Medline]
  16. Kleschyov AL, Muller B, Keravis T, Stoeckel ME, and Stoclet JC. Adventitia-derived nitric oxide in rat aortas exposed to endotoxin: cell origin and functional consequences. Am J Physiol Heart Circ Physiol 279: H2743–H2751, 2000.[Abstract/Free Full Text]
  17. Kleschyov AL, Muller B, Schott C, and Stoclet JC. Role of adventitial nitric oxide in vascular hyporeactivity induced by lipopolysaccharide in rat aorta. Br J Pharmacol 124: 623–626, 1998.[CrossRef][Web of Science][Medline]
  18. Lakshminrusimha S, D'Angelis CA, Russell JA, Nielsen LC, Gugino SF, Nickerson PA, and Steinhorn RH. C-type natriuretic peptide system in fetal ovine pulmonary vasculature. Am J Physiol Lung Cell Mol Physiol 281: L361–L368, 2001.[Abstract/Free Full Text]
  19. MacFarland RT. Molecular aspects of cyclic GMP signaling. Zoological Sci 12: 151–163, 1995.[Web of Science][Medline]
  20. Matoba T and Shimokawa H. Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in animals and humans. J Pharm Sci 92: 1–6, 2003.[CrossRef]
  21. Miura H, Bosnjak JJ, Ning G, Saito T, Miura M, and Gutterman DD. Role for hydrogen peroxide in flow-induced dilation of human coronary arterioles. Circ Res 92: e31–e40, 2003.[Abstract/Free Full Text]
  22. Rey FE, Li XC, Carretero OA, Garvin JL, and Pagano PJ. Perivascular superoxide anion contributes to impairment of endothelium-dependent relaxation: role of gp91(phox). Circulation 106: 2497–2502, 2002.[Abstract/Free Full Text]
  23. Schindler MB, Hislop AA, and Haworth SG. Postnatal changes in response to norepinephrine in the normal and pulmonary hypertensive lung. Am J Respir Crit Care Med 170: 641–646, 2004.[Abstract/Free Full Text]
  24. Schrammel A, Behrends S, Schmidt K, Koesling D, and Mayer B. Characterization of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one as a heme-site inhibitor of nitric oxide-sensitive guanylyl cyclase. Mol Pharmacol 50: 1–5, 1996.[Abstract]
  25. Shimokawa H and Matoba T. Hydrogen peroxide as an endothelium-derived hyperpolarizing factor. Pharmacol Res 49: 543–549, 2004.[CrossRef][Web of Science][Medline]
  26. Spina D, Fernandes LB, Preuss JM, Hay DW, Muccitelli RM, Page CP, and Goldie RG. Evidence that epithelium-dependent relaxation of vascular smooth muscle detected by co-axial bioassays is not attributable to hypoxia. Br J Pharmacol 105: 799–804, 1992.[Web of Science][Medline]
  27. Spina D and Page CP. The release of a non-prostanoid inhibitory factor from rabbit bronchus detected by co-axial bioassay. Br J Pharmacol 102: 896–903, 1991.[Web of Science][Medline]
  28. Steinhorn RH, Morin FC III, Gugino SF, Giese EC, and Russell JA. Developmental differences in endothelium-dependent responses in isolated ovine pulmonary arteries and veins. Am J Physiol Heart Circ Physiol 264: H2162–H2167, 1993.[Abstract/Free Full Text]
  29. Steinhorn RH, Morin FC III, and Russell JA. The adventitia may be a barrier specific to nitric oxide in rabbit pulmonary artery. J Clin Invest 94: 1883–1888, 1994.[Web of Science][Medline]
  30. Steinhorn RH, Russell JA, Lakshminrusimha S, Gugino SF, Black SM, and Fineman JR. Altered endothelium-dependent relaxations in lambs with high pulmonary blood flow and pulmonary hypertension. Am J Physiol Heart Circ Physiol 280: H311–H317, 2001.[Abstract/Free Full Text]
  31. Villamor E, Kessels CG, Fischer MA, Bast A, de Mey JG, and Blanco CE. Role of superoxide anion on basal and stimulated nitric oxide activity in neonatal piglet pulmonary vessels. Pediatr Res 54: 372–381, 2003.[CrossRef][Web of Science][Medline]
  32. Villamor E, Perez Vizcaino F, Tamargo J, and Moro M. Effects of group B Streptococcus on the responses to U46619, endothelin-1, and noradrenaline in isolated pulmonary and mesenteric arteries of piglets. Pediatr Res 40: 827–833, 1996.[Web of Science][Medline]
  33. Villamor E, Perez-Vizcaino F, Cogolludo AL, Conde-Oviedo J, Zaragoza-Arnaez F, Lopez-Lopez JG, and Tamargo J. Relaxant effects of carbon monoxide compared with nitric oxide in pulmonary and systemic vessels of newborn piglets. Pediatr Res 48: 546–553, 2000.[Web of Science][Medline]
  34. Wang H, Pagano P, Du Y, Cayatte A, Quinn M, Brecher P, and Cohen R. Superoxide anion from the adventitia of the rat thoracic aorta inactivates nitric oxide. Circ Res 82: 810–818, 1998.[Abstract/Free Full Text]




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