Endothelium-derived reactive oxygen species and endothelin-1 attenuate NO-dependent pulmonary vasodilation following chronic hypoxia

Nikki L. Jernigan, Benjimen R. Walker, and Thomas C. Resta

Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131-0001

Submitted 16 December 2003 ; accepted in final form 3 June 2004


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Vasodilatory responses to exogenous nitric oxide (NO) are diminished following exposure to chronic hypoxia (CH) in isolated, perfused rat lungs. We hypothesized that both endothelium-derived reactive oxygen species (ROS) and endothelin-1 (ET-1) mediate this attenuated NO-dependent pulmonary vasodilation following CH. To test this hypothesis, we examined vasodilatory and vascular smooth muscle (VSM) Ca2+ responses to the NO donor spermine NONOate in UTP-constricted, isolated pressurized small pulmonary arteries from control and CH rats. Consistent with our previous findings in perfused lungs, we observed attenuated NO-dependent vasodilation following CH in endothelium-intact vessels. However, in endothelium-denuded vessels, responses to spermine NONOate were augmented in CH rats compared with controls, thus demonstrating an inhibitory influence of the endothelium on NO-dependent reactivity following CH. Whereas both the ROS scavenger tiron and the ETA receptor antagonist BQ-123 augmented NO-dependent reactivity in endothelium-intact vessels from CH rats, neither fully restored vasodilatory responses to those observed following endothelium denudation in vessels from CH rats. In contrast, the combination of tiron and BQ-123 or the nonselective ET receptor antagonist PD-145065 enhanced NO responsiveness in endothelium-intact vessels from CH rats similar to that observed following endothelium denudation. We conclude that both endothelium-derived ROS and ET-1 attenuate NO-dependent pulmonary vasodilation following CH. Furthermore, CH augments pulmonary VSM reactivity to NO.

pulmonary hypertension; nitric oxide; isolated small pulmonary arteries; intracellular calcium; vascular smooth muscle


THE DEVELOPMENT OF PULMONARY HYPERTENSION and right ventricular hypertrophy following chronic exposure to hypoxia results from polycythemia, pulmonary arterial remodeling, and arterial constriction. The severity of chronic hypoxia (CH)-induced pulmonary hypertension has been shown to be reduced by the potent vasodilator nitric oxide (NO) (12, 37). NO is produced in vascular endothelial cells by endothelial nitric oxide synthase (eNOS). Several investigators (15, 30, 39) have shown that CH augments pulmonary vasodilatory responses to endothelium-derived nitric oxide (EDNO)-dependent vasodilators. This altered vasoreactivity is associated with increased pulmonary eNOS levels, gene expression, and activity (13, 22, 27, 28, 34, 39). Interestingly, despite enhanced reactivity to EDNO-dependent vasodilators following CH, we have demonstrated impaired responsiveness to exogenous NO in isolated lungs from CH rats compared with controls (16, 28). The mechanism by which CH attenuates vasodilation to exogenous NO has not been clearly defined but may be a function of either decreased vascular smooth muscle sensitivity to NO or altered endothelial regulation of vascular tone.

It is feasible that NO bioinactivation, resulting from increased generation of reactive oxygen species (ROS; 3, 31) or impaired ROS scavenging (26), leads to diminished NO-dependent pulmonary vasoreactivity in CH rats. Considering the prominent role of the endothelium as a source of ROS (38, 42), we hypothesized that endothelium-derived ROS attenuates NO-dependent pulmonary vasodilation following CH.

Alternatively, it is possible that CH-induced attenuation of NO-dependent vasoreactivity is a function of increased pulmonary expression of endothelin-1 (ET-1; 14, 21), since this endothelium-derived vasoconstrictor peptide has been shown to have a direct inhibitory effect on NO-mediated dilation (2). Therefore, we further hypothesized that ET-1 contributes to diminished NO-dependent pulmonary vasodilation following CH. To test these hypotheses, we assessed exogenous NO-dependent vasodilatory responses and vascular smooth muscle (VSM) intracellular calcium concentrations ([Ca2+]i) in endothelium-intact and endothelium-denuded small pulmonary arteries from control and CH rats. Parallel experiments were conducted in the presence of the ROS scavenger 4,5-dihydroxy-1,3-benzene-disulfonic acid (tiron) to examine the effect of endogenous ROS on NO-dependent reactivity. Furthermore, NO-dependent pulmonary vasodilation was assessed in the presence of the selective ETA receptor antagonist BQ-123 or the nonselective ET receptor antagonist PD-145065. Our findings suggest that both ROS and ET-1 contribute to diminished pulmonary vasodilation to exogenous NO following CH.


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All protocols and surgical procedures employed in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the University Of New Mexico School Of Medicine (Albuquerque, NM).

Experimental Groups

Male Sprague-Dawley rats (200–250 g, Harlan Industries) were divided into two groups for each experiment. Animals designated for exposure to CH were housed in a hypobaric chamber with barometric pressure maintained at ~380 mmHg for 4 wk. The chamber was opened three times per week to provide animals with fresh food, water, and clean bedding. On the day of experimentation, rats were removed from the hypobaric chamber and immediately placed in a Plexiglas chamber continuously flushed with a 12% O2-88% N2 gas mixture to reproduce inspired PO2 (~70 mmHg) within the hypobaric chamber. Age-matched control animals were housed at ambient barometric pressure (~630 mmHg). All animals were maintained on a 12:12-h light-dark cycle.

Cannulation of Small Pulmonary Arteries for Dimensional Analysis

Rats were anesthetized with pentobarbital sodium (32.5 mg ip), and the heart and lungs were exposed by midline thoracotomy. The left lung was removed and immediately placed in ice-cold physiological saline solution (PSS) containing (in mM) 129.8 NaCl, 5.4 KCl, 0.83 MgSO4, 19 NaHCO3, 1.8 CaCl2, and 5.5 glucose. The lung was pinned out in iced PSS in a Silastic-coated dissection dish. A fourth-order intrapulmonary artery [150–300 µm inner diameter (ID)] of ~1 mm length and without branches was dissected free and transferred to a vessel chamber (Living Systems, CH-1) containing aerated PSS. The proximal end of the artery was cannulated with a tapered glass pipette, secured in place with a single strand of silk ligature, and gently flushed to remove any blood from the lumen. Next, the distal end of the vessel was cannulated, and the artery was stretched longitudinally to approximate its in vivo length and then pressurized to 12 mmHg. The vessel chamber was transferred to the stage of a Nikon Eclipse TS100 microscope, and the preparation was superfused with PSS equilibrated with 10% O2-6% CO2-balance N2 (PO2 ~56 Torr). A vessel chamber cover was positioned to permit this same gas mixture to flow over the top of the chamber bath. PO2, PCO2, and pH of the superfusate were monitored with a blood-gas analyzer (Radiometer). Bright-field images of vessels were obtained with an IonOptix CCD100M camera, and dimension analysis was performed by IonOptix Sarclen software to measure internal diameter. We have used similar methods in recent studies from our laboratory (8).

Measurement of VSM Calcium

Pressurized arteries were loaded via the adventitial surface with the cell-permeant, ratiometric, Ca2+-sensitive fluorescence indicator fura-2 AM (2 µM) (Molecular Probes) for 45 min at room temperature in the dark. Administration of fura-2 AM to the abluminal surfaces of pressurized resistance arteries has been shown to preferentially load VSM cells (20). The diluted fura-2 AM solution was equilibrated with the 10% O2 gas mixture during the loading period. Vessels were rinsed for 20 min with warmed, aerated PSS following the loading period to wash out excess dye and to allow for hydrolysis of AM groups by intracellular esterases. Fura-loaded vessels were alternatively excited at 340 and 380 nm at a frequency of 10 Hz with an IonOptix Hyperswitch dual excitation light source, and the respective 510-nm emissions were collected with a photomultiplier tube. All experiments were completed using the same exposure times for the 340- and 380-nm wavelengths in both groups. Background-subtracted 340-nm/380-nm fluorescence emission ratios (F340/F380) were calculated with IonOptix Ion Wizard software and recorded continuously throughout the experiment, with simultaneous measurement of ID from bright-field images as described above. These methods for simultaneous dimensional analysis and measurement of VSM [Ca2+]i are similar to those previously described by our laboratory for mesenteric resistance arteries (8).

Verification of Endothelial Integrity/Disruption

For experiments in endothelium-intact vessels, we assessed endothelial integrity before experimentation by preconstricting arteries with UTP (~30% of baseline ID) and measuring the subsequent vasodilatory response to ACh (1 µM). ACh mediates dose-dependent vasodilation and decreases in VSM [Ca2+]i in this preparation as determined in preliminary experiments from our laboratory. In some experiments, the vessel lumen was rubbed with a strand of moose mane to disrupt the endothelium following cannulation of the proximal end of the artery. The effectiveness of endothelial disruption was verified by the lack of a vasodilatory response to ACh (1 µM) in UTP-constricted vessels. After administration of ACh, vessels were rinsed in normal PSS and loaded with fura-2 AM as above.

Isolated Vessel Experiments

Vasodilatory responses and changes in VSM calcium to spermine NONOate in endothelium-intact and endothelium-denuded small pulmonary arteries. To examine the influence of endothelial vasoactive factors on exogenous NO-mediated vasodilation, we assessed responses to the NO donor spermine NONOate in endothelium-intact and endothelium-denuded small pulmonary arteries from control and CH rats at 12 mmHg and 10% O2. Endothelium-intact arteries in this experiment and the following experiments were treated with N{omega}-nitro-L-arginine (L-NNA, 300 µM) to minimize potential complicating influences of endogenous NO on the observed responses. After fura-2 AM loading and 20 min of equilibration, vessels from either group were preconstricted (~ 30% of baseline ID) with UTP-Na3 (2.5–5 µM). UTP provides consistent and stable vasoconstrictor responses in this preparation. UTP constriction was allowed to stabilize before a cumulative concentration-response relationship to spermine NONOate (10–9–10–5 M) was determined in these vessels.

Effect of ROS scavenging on responses to spermine NONOate. The contribution of oxygen radicals in altering vasodilatory responses to NO in each group of rats was determined with the membrane-permeable ROS scavenger tiron. Tiron (10 mM) was added to the superfusate immediately after the second equilibration and was present throughout the experiment. We have previously used this dose of tiron to scavenge oxygen radicals in perfused lungs and have recently demonstrated the ability of tiron to inhibit xanthine/xanthine oxidase-induced ROS production in isolated small pulmonary arteries from control animals (17). Responses to spermine NONOate (10–9–10–5 M) were then determined in endothelium-intact and endothelium-denuded small pulmonary arteries from control and CH rats.

Effect of ET receptor inhibition on responses to spermine NONOate. The contribution of ET-1-mediated stimulation of VSM ET receptors in altering vasodilatory responses to NO in each group of rats was determined with the selective ETA receptor antagonist BQ-123 or the nonselective ET receptor antagonist PD-145065. BQ-123 (10 µM) or PD-145065 (1 µM) was added to the superfusate immediately after the second equilibration and was present throughout the experiment. Responses to spermine NONOate (10–9–10–5 M) were then determined in endothelium-intact small pulmonary arteries from control and CH rats as above. To establish the efficacy of BQ-123 and PD-145065 in this preparation, we generated a cumulative concentration response relationship for ET-1 and the ETB receptor agonist IRL-1620 in separate sets of isolated small pulmonary arteries from control rats in the absence or presence of BQ-123 (10 µM) or PD-145065 (1 µM).

Effect of combined ROS scavenging and ET receptor inhibition on responses to spermine NONOate. To determine the collective contribution of endogenous ROS and ET-1 on NO-dependent vasodilatory responses, we added either tiron (10 mM) and BQ-123 (10 µM) or tiron (10 mM) and PD-145065 (1 µM) to the superfusate immediately after the second equilibration, and they were present throughout the experiment. Responses to spermine NONOate (10–9–10–5 M) were then determined in endothelium-intact small pulmonary arteries from control and CH rats as above.

Calculations and Statistics

Vasodilatory responses were calculated as a percent reversal of UTP-induced vasoconstriction. All data are expressed as means ± SE, and values of n refer to the number of animals in each group. Two-way ANOVA was used to make comparisons. If differences were detected, individual groups were compared with the Student-Newman-Keuls test. A probability of P ≤ 0.05 was accepted as significant for all comparisons.


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Vasodilatory Responses and Changes in VSM Calcium to Spermine NONOate in Endothelium-Intact and Endothelium-Denuded Small Pulmonary Arteries

Figure 1A depicts a representative trace of vessel ID and VSM [Ca2+]i in response to UTP and the subsequent addition of spermine NONOate (10–6 M) in an endothelium-intact control artery. We observed only modest decreases in [Ca2+]i associated with a substantial spermine NONOate-induced dilation (~70%). In contrast, the L-type calcium channel inhibitor diltiazem (50 µM) resulted in the expected decrease in VSM [Ca2+]i in a KCl-constricted artery (Fig. 1B), demonstrating our ability to assess agonist-induced changes in [Ca2+]i in this preparation.



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Fig. 1. Representative traces of inner diameter (ID) and vascular smooth muscle (VSM) intracellular calcium concentration ([Ca2+]i, expressed as F340/F380) in endothelium-intact control arteries for spermine NONOate (10–6 M) reversal of UTP (5 µM final concentration) preconstriction (A) and diltiazem (50 µM) reversal of KCl (50 mM) preconstriction (B). {Delta}, change in.

 
L-NNA produced modest vasoconstriction in endothelium-intact CH vessels, but not controls (Table 1). This L-NNA-induced tone was added to the UTP constriction to match the final level of preconstriction (~30% of baseline ID) between groups. Tiron, BQ-123, and PD-145065 did not further alter VSM tone. Vasodilatory responses to spermine NONOate were attenuated in endothelium-intact vessels from CH rats vs. controls (Fig. 2A), as previously observed in isolated saline-perfused lungs (16). In contrast, following disruption of the endothelium, vasodilatory responses to spermine NONOate in CH arteries were augmented compared with both endothelium-denuded control vessels and CH intact vessels (Fig. 2A). Endothelial denudation had no significant effect on vasodilatory responses in vessels from control animals. These findings support a role for endothelial factors to attenuate NO-dependent vasodilation in arteries from CH rats, but not controls.


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Table 1. Change in inner diameter (percent baseline) in endothelium-intact vessels following administration of L-NNA plus vehicle, tiron, BQ-123, or PD-145065

 


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Fig. 2. A: vasodilatory responses (% reversal of UTP-induced vasoconstriction). B: changes in VSM [Ca2+]i to spermine NONOate (10–9–10–5 M) in endothelium-intact [Endo (+)] and -denuded [Endo (–)] small pulmonary arteries from control (n = 6/group) and chronically hypoxic (CH, n = 6/group) rats. Experiments with endothelium-intact arteries were conducted in the presence of N{omega}-nitro-L-arginine (L-NNA, 300 µM). Values are means ± SE. *P ≤ 0.05 vs. corresponding control group; #P < 0.05 vs. CH Endo (+).

 
Baseline F340/F380 ratios were not different between groups (0.90 ± 0.02 for control and 0.85 ± 0.03 for CH; n = 53 each group), and UTP induced similar increases in ratios between groups ({Delta} in ratio of 0.08 ± 0.03 for control and 0.07 ± 0.02 for CH). These modest ratio changes in response to UTP reflect a major contribution of VSM Ca2+ sensitization mechanisms in UTP-induced vasoconstriction as demonstrated in preliminary studies (19). Spermine NONOate-induced decreases in VSM [Ca2+]i were diminished in CH vs. control vessels (Fig. 2B). There were no differences in VSM [Ca2+]i changes in endothelium-intact vs. -denuded vessels in either group. These data suggest that NO-mediated vasodilation in pulmonary arteries from CH rats is less dependent on decreases in VSM [Ca2+]i compared with control vessels.

Effect of ROS Scavenging on Responses to Spermine NONOate

The ROS scavenger tiron significantly augmented vasodilatory responses to spermine NONOate (10–7–10–5 M) in endothelium-intact arteries from CH rats (Fig. 3A) compared with untreated arteries from the same group (Fig. 2A), but was without effect in endothelium-intact control vessels (Fig. 3A). Furthermore, pretreatment with tiron normalized responses between control and CH groups. However, the ROS scavenger did not restore reactivity in endothelium-intact arteries from CH rats to that observed in CH denuded vessels (Fig. 3A), suggesting that other endothelium-derived factors may contribute to attenuated NO-dependent vasoreactivity following CH. In contrast to endothelium-intact vessels, no differences were observed between endothelium-denuded arteries treated with tiron (Fig. 3B) vs. vehicle (Fig. 2A) in either group. Changes in vessel wall calcium were consistent with Fig. 2B and are therefore not illustrated for these or the remainder of experiments.



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Fig. 3. Vasodilatory responses to spermine NONOate (10–9–10–5 M) in the presence of tiron (10 mM) in endothelium-intact (A) and endothelium-denuded small pulmonary arteries (B) from control (n = 5/group) and CH (n = 5/group) rats. Experiments with endothelium-intact vessels were conducted in the presence L-NNA (300 µM). Values are means ± SE. *P ≤ 0.05 vs. Control Endo (–) + Tiron; #P ≤ 0.05 vs. Endo (–) CH from Fig. 2A; {tau}P ≤ 0.05 vs. Endo (+) CH from Fig. 2A.

 
Effect of ET Receptor Inhibition on Responses to Spermine NONOate

Both the selective ETA receptor inhibitor BQ-123 and the nonselective ET receptor antagonist PD-145065 diminished ET-1-dependent vasoconstriction in endothelium-intact control arteries, with PD-145065 having a significantly greater inhibitory influence than BQ-123 at 10–8 and 10–9 M concentrations of ET-1 (Fig. 4A). Furthermore, PD-145065 largely attenuated the concentration-response relationship to IRL-1620, illustrating the ability of PD-145065 to inhibit ETB-receptor mediated vasoconstriction (Fig. 4B).



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Fig. 4. Vasoconstriction (percent of baseline ID) to endothelin-1 (10–12–10–7 M, A) in the absence or presence of BQ-123 (10 µM) or PD-145065 (1 µM) and IRL-1620 (10–10–10–8 M, B) in the absence or presence of PD-145065 (1 µM) in endothelium-intact small pulmonary arteries from control (n = 4–5/group) rats. Experiments were conducted in the presence of L-NNA (300 µM). Values are means ± SE. *P < 0.05 vs. vehicle and #P < 0.05 vs. BQ-123 treated.

 
Pretreatment with BQ-123 significantly augmented vasodilatory responses to spermine NONOate (10–7–10–5 M) in endothelium-intact arteries from CH rats (Fig. 5A) compared with untreated arteries from the same group (Fig. 2A), but was without a significant effect in endothelium-intact control vessels. Furthermore, NO-dependent reactivity was greater in CH arteries vs. controls following administration of BQ-123 at all concentrations of spermine NONOate above 10–9 M. Although administration of BQ-123 to endothelium-intact vessels from CH rats (Fig. 5A) did not normalize responses to those of endothelium-denuded CH arteries (Fig. 2A) at lower concentrations of spermine NONOate (10–9–10–8 M, Fig. 5A), responses were normalized between these groups at higher concentrations of the NO donor (10–7–10–5 M).



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Fig. 5. Vasodilatory responses to spermine NONOate (10–9–10–5 M) in endothelium-intact small pulmonary arteries from control (n = 5) and CH (n = 5) rats in the presence of BQ-123 (10 µM, A) or PD-145065 (1 µM, B). Experiments were conducted in the presence of L-NNA (300 µM). Values are means ± SE. *P < 0.05 vs. Control + BQ-123 (A) or Control + PD-145065 (B); #P < 0.05 vs. Endo (–) CH from Fig. 2A; {tau}P < 0.05 vs. Endo (+) CH from Fig. 2A.

 
Similarly, pretreatment with PD-145065 yielded greater vasodilatory responses to spermine NONOate in endothelium-intact arteries from CH rats compared with controls (Fig. 5B) and vs. untreated CH vessels from Fig. 2A. However, in contrast to effects of BQ-123, PD-145065 fully normalized responses in endothelium-intact arteries from CH rats (Fig. 5B) to those of denuded CH vessels (Fig. 2A).

Effect of Combined ROS Scavenging and ET Receptor Inhibition on Responses to Spermine NONOate

Pretreatment with both tiron and BQ-123 significantly augmented vasodilatory responses in endothelium-intact arteries from CH rats (Fig. 6A) vs. untreated vessels from the same group (Fig. 2A). Furthermore, this combination of ROS and ETA-receptor inhibition fully restored reactivity in CH arteries (Fig. 6A) to that observed in denuded vessels from CH animals (Fig. 2A). However, pretreatment with tiron and BQ-123 had no significant effect on responses from control vessels. Consequently, NO-mediated responses were greater in arteries from CH vs. control rats in the presence of tiron + BQ-123 (Fig. 6A).



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Fig. 6. Vasodilatory responses to spermine NONOate (10–9–10–5 M) in endothelium-intact small pulmonary arteries from control (n = 5) and CH (n = 5) rats in the presence of BQ-123 (10 µM) and tiron (10 mM) (A) or PD-145065 (1 µM) and tiron (10 mM) (B). Experiments were conducted in the presence of L-NNA (300 µM). Values are means ± SE. *P < 0.05 vs. Control + BQ-123 and tiron (A) or PD-145065 and tiron (B); {tau}P < 0.05 vs. Endo (+) CH from Fig. 2A.

 
The combination of PD-145065 and tiron normalized vasodilatory responses to NO in endothelium-intact CH vessels (Fig. 6B) to those demonstrated in endothelium-disrupted arteries from CH rats (Fig. 2A), similar to that observed with PD-145065 alone (Fig. 5B). Therefore, in contrast to effects of tiron to augment NO-dependent responsiveness in BQ-123-treated arteries from CH rats (Fig. 6A), there was no additional effect of tiron to enhance NO reactivity when coadministered with PD-145065 (Fig. 6B).


    DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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The present study examined the role of endothelium-derived ROS and ET-1 in attenuated NO-dependent pulmonary vasodilation following CH. The major findings from this study are 1) vasodilatory responses to the NO-donor spermine NONOate were attenuated in endothelium-intact small pulmonary arteries from CH rats compared with controls; 2) after endothelium-denudation, vasodilatory responses to spermine NONOate were augmented following CH, whereas NO-mediated decreases in VSM [Ca2+]i were diminished compared with control arteries; 3) the ROS scavenger tiron improved vasodilation in endothelium-intact vessels from CH rats but was without effect in endothelium-intact control vessels or endothelium-denuded vessels from either group; 4) the selective ETA receptor antagonist BQ-123 augmented vasodilatory responses in endothelium-intact vessels from CH rats but not controls; and 5) the combination of tiron and BQ-123 or the nonselective ET receptor antagonist PD-145065 normalized vasodilatory responses in endothelium-intact vessels to responses in endothelium-denuded vessels following CH. These data suggest the attenuated pulmonary vasodilatory response to NO following CH is mediated by release of the endothelial vasoactive factors ROS and ET-1. Furthermore, CH enhances pulmonary VSM reactivity to NO but may alter NO-mediated regulation of intracellular Ca2+ handling.

Although some investigators have shown that rats that were returned to normoxia following prolonged hypoxia demonstrate impaired EDNO-mediated vasodilation (1, 10), several others, including our laboratory, have observed augmented EDNO-dependent pulmonary vasodilation following CH (15, 2730, 39). This response is associated with upregulation of pulmonary eNOS mRNA and protein levels (13, 22, 27, 28, 34, 39) as well as increased synthesis of NO (13, 15, 25, 39). Despite this enhanced reactivity to EDNO-mediated agonists, studies from our laboratory (16, 28) have recently reported that CH attenuates vasodilatory responses to exogenous NO in isolated, saline-perfused rat lungs. However, the mechanism of this attenuated NO-dependent vasoreactivity following CH is not fully understood.

It is possible that the attenuated vasodilatory response following CH is a result of diminished VSM sensitivity to NO. Therefore, the augmented response to endogenously produced NO may be a result of greater NO production masking decreased VSM sensitivity to NO following CH. Our laboratory (16, 18) has recently examined potential alterations in VSM sensitivity to the NO/cGMP pathway as an explanation for the attenuated vasodilatory responsiveness to exogenous NO. In these studies, we observed no significant differences in soluble guanylyl cyclase protein levels or cGMP levels in lungs from CH rats compared with control rats. Although phosphodiesterase-5 (PDE-5) inhibitors greatly augmented vasodilatory responses to S-nitroso-N-acetyl penicillamine and authentic NO in lungs from CH rats, indicating increased cGMP degradation by PDE-5, the attenuated vasodilatory response to NO in CH lungs persisted. Moreover, we demonstrated a marked increase in cGMP-dependent protein kinase (PKG) expression and activity following CH (18). Together these data suggest that the NO-signal transduction pathway is not compromised following CH. Indeed, our current observations that endothelial disruption revealed an enhanced NO-responsiveness in CH arteries compared with controls indicate that VSM sensitivity to NO is enhanced in response to hypoxic acclimation. These findings further suggest that endothelial factors attenuate NO-dependent reactivity following CH.

One possible explanation for the attenuated NO-mediated vasodilation following CH in endothelium-intact vessels may involve a decrease in NO bioavailability due to increased ROS production (3, 31). Several studies, including studies from our laboratory, have demonstrated the ability of antioxidants to improve NO-dependent vasodilatory responses following CH (6, 17, 40). Corresponding to diminished NO-dependent vasoreactivity, we found that ROS levels were greater in L-NNA-treated normoxic pulmonary arteries from CH rats compared with controls, suggesting that elevated vascular production of ROS may contribute to this altered vasoresponsiveness. Consistent with this previous study, our current findings that tiron normalized vasodilatory responses in endothelium-intact vessels from CH rats to those observed in vessels from control animals strongly support a role for ROS in attenuating NO-dependent vasoreactivity following CH.

It is possible increased ROS production is a result of reoxygenation following hypoxia. Indeed, we have previously demonstrated that hypoxic ventilation increases and normalizes vasodilatory responses to NO between groups and decreases ROS levels, signifying a dependence on the availability of O2 (17). Alternatively, CH-induced ROS synthesis could result from increased activity of ROS-generating enzymes or rather compromised mechanisms of ROS scavenging (6, 26). Consistent with this possibility, Brennan et al. (6) has recently shown a reduction in pulmonary arterial SOD activity as well as elevated NADPH oxidase expression corresponding to increased ROS production and diminished NO-dependent vasoreactivity in pulmonary hypertensive fetal lambs. Potentially, CH has similar effects to increase pulmonary arterial ROS production.

Interestingly, tiron was without effect in endothelium-denuded vessels from either group, suggesting ROS-induced attenuation of NO-mediated vasodilation is endothelium-dependent. Together these data support a role for endothelium-derived ROS in the diminished NO-mediated vasodilation following CH. Although pretreatment with tiron improved vasodilatory responses in intact vessels from CH rats, these responses remained diminished compared with endothelium-denuded vessels from CH rats, suggesting some other endothelium-derived vasoactive factor contributes to the diminished vasodilatory response to NO following CH.

The potent vasoconstrictor ET-1 is synthesized and released from pulmonary endothelial cells and mediates increases in pulmonary vascular resistance through activation of ETA and ETB receptors on pulmonary VSM (5, 24). Prolonged exposure to hypoxia increases ET-1 mRNA and protein, circulating ET-1 plasma levels (9, 11), and expression of ETA and ETB receptors (23). Therefore, it is additionally possible that enhanced production of ET-1 and increased receptor activity following CH acts to hinder NO-induced vasodilation. In the current study, the addition of the ETA receptor antagonist BQ-123 greatly augmented vasodilatory responses to spermine NONOate following CH, suggesting ET-1 inhibits NO-mediated vasodilation. Although we have presently demonstrated an effect of BQ-123 to augment vasodilatory responses to NO in endothelium-intact vessels from CH rats, these responses remained diminished compared with endothelium-denuded vessels from CH rats at the lower doses of spermine NONOate (10–9–10–8 M). However, the addition of tiron and BQ-123 normalized responses in endothelium-intact vessels to those of endothelium-denuded vessels from CH rats, suggesting that both endothelium-derived ROS and ET-1 oppose NO-mediated vasodilation.

Considering that pulmonary arterial ETB receptor expression is upregulated in response to hypoxic acclimation (36) and may thus contribute to ET-1-dependent vasoconstriction in this vascular bed, additional experiments were performed to compare effects of the nonselective ET receptor antagonist PD-145065 on NO-mediated vasodilation in vessels from each group of rats. Interestingly, PD-145065 markedly enhanced NO-dependent reactivity in arteries from CH rats and fully normalized responses to those observed in endothelium-denuded vessels from the same group. Furthermore, in contrast to BQ-123-treated vessels, tiron provided no additional effect to augment responses to NO in CH arteries pretreated with PD-145065. One possible explanation for these disparate effects of tiron in BQ-123- vs. PD-145065-treated arteries is that endogenous ET-1 induces ROS formation via stimulation of endothelial ETB receptors in arteries from CH rats. Indeed, ET-1 has previously been demonstrated to increase ROS-induced proliferation of cultured ovine pulmonary arterial smooth muscle (41). Considering that both pretreatment with BQ-123 + tiron and PD-145065 in endothelium-intact arteries augmented NO-dependent vasodilation similar to endothelium-denuded vessels in the CH group, we feel that an endothelial source of ET-1 most likely explains the inhibitory influence of the endothelium on NO reactivity in these vessels. However, we cannot rule out a potential role for VSM-derived ET-1 in the observed effects of ET receptor blockade. Although it is beyond the focus of the current study, further investigation is needed to examine the putative contribution of ETB-mediated ROS production in attenuated NO-dependent pulmonary vasodilation following CH.

Whereas enhanced pulmonary VSM reactivity to NO following CH may result in part from greater PKG-1 expression and activity as we have recently reported (18), it is evident from the current study that this upregulation of PKG-1 does not correlate with a greater decrease in VSM [Ca2+]i. Rather, augmented VSM sensitivity to NO following hypoxic exposure is paradoxically associated with a diminished fall in VSM [Ca2+]i despite no significant differences in either baseline or UTP-mediated increases in F340/F380 ratios between control and CH arteries. These results are in apparent contrast to earlier studies demonstrating elevated basal VSM [Ca2+]i (4, 35) in pulmonary myocytes from CH rats vs. controls. Although the reason for this discrepancy is unclear, it is possible that measurements in isolated myocytes from larger pulmonary arteries do not reflect VSM [Ca2+]i levels in the smaller vessels used in the current study. Together, our present findings suggest a unique effect of CH to impair NO-mediated regulation of intracellular Ca2+ handling and to mediate a compensatory increase in NO-dependent desensitization of the contractile apparatus to Ca2+. Although beyond the focus of the current investigation, challenges of future studies will be to define the mechanisms responsible for this divergence in VSM NO signal transduction between pulmonary arteries from control and CH rats, including a potential role for CH to enhance PKG-mediated inhibition of Rho-kinase-induced Ca2+ sensitization (32, 33) and to interfere with regulation of Ca2+ influx and sequestration mechanisms by PKG (7).

In conclusion, the present study suggests that endothelium-derived ROS and ET-1 both contribute to diminished NO-dependent pulmonary vasodilation following CH. Furthermore, VSM sensitivity to NO is enhanced following CH, a response that reflects a switch in NO signaling toward mechanisms of myofilament Ca2+ desensitization.


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 ABSTRACT
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This work was supported by a Scientist Development Grant from the American Heart Association (T. C. Resta), by National Institutes of Health Grants RR-16480 (T. C. Resta), HL-58124 and HL-63207 (B. R. Walker), and by a Parker B. Francis Fellowship in Pulmonary Research (T. C. Resta).


    ACKNOWLEDGMENTS
 
The authors thank Pam Allgood for technical assistance.

Present address for N. L. Jernigan: Univ. of Mississippi Medical Center, Dept. of Physiology and Biophysics, 2500 N. State St., Jackson, MS 39216-4505.


    FOOTNOTES
 

Address for reprint requests and other correspondence: T. C. Resta, Dept. of Cell Biology and Physiology, MSC08 4750, 1 Univ. of New Mexico, Albuquerque, NM 87131-0001 (E-mail: tresta{at}salud.unm.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
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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