Hypoxia

Study objective: To investigate the effects of long-term nitric oxide (NO) inhalation on the recovery process of right ventricular hypertrophy (RVH) and functional alterations in the NO-cyclic guanosine monophosphate (cGMP) relaxation pathway in rat conduit pulmonary arteries (PAs) in established chronic hypoxic pulmonary hypertension.

Materials and methods: A total of 35 rats were exposed to chronic hypobaric hypoxia (380 mm Hg, 10% oxygen), and 39 rats were exposed to air for 10 days. Both groups were then exposed to 3 or 10 days of NO 10 ppm, NO 40 ppm, or air (control groups for each NO concentration), resulting in a total of 16 groups. Acetyleholine- and sodium nitroprusside (SNP)-induced relaxation were evaluated in precontracted PA rings. RVH was assessed by heart weight ratio of fight ventricle to left ventricle plus septum.

Results: NO inhalation had no effect on either the regression of RVH or the recovery process of impaired relaxation induced by acetyleholine or SNP in a endothelium-intact hypertensive conduit extrapulmonary artery or intrapulmonary artery (IPA). In a normal endothelium-intact conduit IPA, 40 ppm NO inhalation for 10 days partially augmented SNP-indueed relaxation, but not that induced by acetylcholine.

Conclusion: Continuous NO inhalation did not affect the regression process of either established RVH or the impaired endogenous NO-cGMP relaxation cascade in a conduit PA in rats during the recovery period after chronic hypoxia.

Key words: acetylcholine; hypoxia; nitric oxide; pulmonary hypertension; recovery; right ventricular hypertrophy; sodium nitroprusside; vasodilation

Abbreviations: cGMP = cyclic guanosine monophosphate; CH = chronic hypoxia; EPA = extrapulmonary artery; IPA = intrapulmonary artery; LV+S = left ventricle plus septum; NO = nitric oxide; NOS = nitric oxide synthase; PA = pulmonary artery; PD[E.sub.5] = phosphodiesterase 5; PG[F.sub.2][alpha] = prostaglandin [F.sub.2][alpha]; PH = pulmonary hypertension; RV fight ventricle; RVH = nght ventricular hypertrophy; SNP = sodium nitroprusside

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The inhalation of nitric oxide (NO) causes acute selective pulmonary vasodilation in patients with pulmonary hypertension (PH), such as COPD, (1) pulmonary fibrosis, (2) ARDS, (3) congenital heart disease with PH, (4) and persistent PH of the neonate. (5) A rebound increase in pulmonary arterial pressure after cessation of NO inhalation is observed in patients with these diseases. (5,6) Long-term NO inhalation therapy has been performed in patients with primary PH (7-9) and idiopathic pulmonary fibrosis. (10) In these patients, exercise tolerance improved during long-term NO inhalation, while it became deteriorated after cessation of the NO inhalation. Since endogenous NO production is impaired after prolonged NO administration, (11,12) prolonged NO inhalation might also impair the endogenous NO production in the pulmonary vasculature of PH.

Endothelial function is impaired in PAs in patients with COPD (13) and rats with chronic hypoxia (GH). (14-16) CH exposure produees chronic PH and pulmonary vascular changes. Previous studies (14,15) have shown that CH PH causes impaired acetylcholine- and sodium nitroprusside (SNP)-induced vasorelaxation in conduit PAs, suggesting impairment of NO-cyclic guanosine monophosphate (cGMP) relaxation pathway. These functional changes and vascular structural changes partly regress during the recovery from GH. (14,17) Although prolonged NO inhalation fails to regress hypoxic vascular structural changes, (18) the effect of prolonged NO inhalation on the recovery process of functional impairment of the pulmonary vasculature is undetermined. In isolated rat lung from CH PH, long-term NO inhalation caused impairment of endothelium-dependent vasorelaxation. (19) If endogenous NO production is impaired by exogenous NO, long-term NO inhalation during the recovery period might deteriorate the recovery process of vasoreactivity in the chronic hypertensive pulmonary arteries (PAs).

The present study was designed to examine whether long-term NO inhalation (10 ppm or 40 ppm) affected the functional recovery process of the impaired NO-cGMP pathway in established hypoxic PH during recovery periods of 3 days or 10 days in room air after exposure to hypobarie hypoxia (880 mm Hg for 10 days). We used acetylcholine as an endothelium-dependent vasodilator and SNP to assess endothelium-independent, cGMP-dependent relaxation.

MATERIALS AND METHODS

Experimental Design

10 ppm NO Study: Male Wistar rats (SLC; Shizuoka, Japan), weighing 170 to 220 g at the beginning of the experiments, were exposed to CH (air at 380 mm Hg) for 10 days as previously described, (14) or kept in room air (normal) for 10 days. Each CH and normal rat was randomly assigned to one of eight groups (four groups for 4-day recovery, four groups for 10-day recovery): CH rats subjected to recovery in room air for 3 days (n = 4) and 10 days (n = 4) [CH control], CH rats subjected to recovery in room air with 10 ppm NO for 3 days (CH/10 ppm/3 day, n = 4) and 10 days (CH/10 ppm/10 day, n = 4), normal rats kept in room air for 3 days (n = 4) and 10 days (n = 4) [normal control], and normal rats kept in room air with 10 ppm NO for 3 days (normal/i0 ppm/3 day, n = 4) and 10 days (normal/10 ppm/10 day, n = 4].

< 40 pprn NO Study: To study 40 ppm NO inhalation, another set of rats were assigned to one of the same eight groups in the 10-ppm study (n = 5, 5, 5, 4, 6, 6, 5, and 6, respectively).

NO Exposure Chamber

After 10 days of exposure to hypoxia, CH rats as well as age-matched normal rats were transferred to glass exposure chambers as previously described, (18,20) and received standard rat chow and water ad libitum for the duration of NO inhalation. Briefly, 10,000 ppm of NO was diluted with fresh air gas in the chamber to obtain 10 ppm and 40 ppm NO concentrations.

Measurement of Urine N[O.sub.3]

To confirm the effectiveness of NO supplementation in our system, urine N[O.sub.3] levels were determined in rats exposed to 40 ppm NO inhalation. Urine samples were analyzed by high-performance liquid chromatography (UV analyzer SPD-10A; Integrator C-R5A; Shimadzu; Kyoto, Japan) at least 3 days after the beginning of continuous NO exposure.

Preparation of PAs

At the end of the recovery period with or without NO inhalation, rats were anesthetized by intraperitoneally administrating pentobarbital (50 mg/kg). Exsangnination was induced by making an incision on the left atrium. After euthanization was established, the lung and heart were removed en bloc and placed in modified Krebs-Henseleit solution at room temperature. The right ventricle (RV) of the heart was dissected from the left ventricle plus septum (LV+S), and these cardiac portions were weighed separately. The value of RV/(LV + S) was then calculated to determine whether right ventricular hypertrophy (RVH) had developed. Two PA segments, a left extrapulmonary artery (EPA) [outside diameter, 1.4 to 1.6 mm] and an intrapulmonary artery (IPA) [0.7- to 1.1-mm outside diameter] were isolated and carefully cleaned of fat and connective tissue. Ring segments (2 mm) were cut (one to two rings from the EPA, and two to four rings from the IPA) and suspended vertically between hooks in an organ bath (20 mL) containing modified Krebs-Henseleit solution, maintained at 37[degrees]C, and bubbled with a mixture of 95% air/5% C[O.sub.2]. The optimal resting tension for the vasodilatory studies was adjusted to 0.75 g for EPA rings and 0.5 g for IPA rings from the normoxic rats, and 1.0 and 0.75 g for the hypoxic rat preparations as previously described. (14) At these resting tensions, the peak contraction was obtained in response to 70 mmol/L KCl. In all experiments, changes in isometric force were measured with a force-displacement transducer (TB-651T; Nihon Kohden; Tokyo, Japan) connected to a carrier amplifier (EF601G; Nihon Kohden) and were recorded on a pen recorder (WT-645G; Nihon Kohden). In some strips, the endothelium was removed by gently rubbing the luminal surface with a small, roughened, stainless steel needle. Successful removal of the endothelium from the PA was verified by the absence of relaxation to ace@choline ([10.sup.-6] mol/L) and by scanning electron microscopy.

Vasodilatation Studies

Arterial rings under an optimal resting tension were washed every 15 to 20 rain and allowed to equilibrate for 120 min. After the equilibration period, 70 mmol/L KCl contraction curves were twice routinely recorded as a measure of maximal contractility, with the contraction shown by the second curve considered as the maximal response. A cumulative concentration response curve to prostaglandin [F.sub.2][alpha] (PG[F.sub.2][alpha]) [[10.sup.-8] to [10.sup.-5] mol/L] was obtained, and the approximate concentration required to produce 50% of the maximal contraction induced by 70 mmol/L KCl was determined. The PA rings were then washed every 15 to 20 rain, allowed to equilibrate for 60 min, and precontracted with PG[F.sub.2][alpha] (3 x [10.sup.-6] to [10.sup.-5] mol/L) to obtain 50 to 70% of the maximal contraction induced by 70 mmol/L KCl. After precontraction with PG[F.sub.2][alpha], cumulative dose-tension curves were obtained for acetylcholine ([10.sup.-8] to [10.sup.-4] mol/L) in the presence of [10.sup.-6] mol/L indomethacin. Finally, [10.sup.-4] mol/L papaverine was added to produce maximal relaxation. With most preparations, this procedure was then repeated using a second vasodilator, SNP ([10.sup.-9] to [10.sup.-5] mol/L). Papaverine-induced relaxation was taken as 100%, and the results of each drug were expressed as a percentage of this relaxation. To further study the smooth-muscle sensitivity to SNP and 8-bromo-cGMP, we analyzed relaxation in response to cumulative concentrations of SNP ([10.sup.-9] to [0.sup.-5] mol/L) and 8-bromo-cGMP ([10.sup.-6] to 3 x [10.sup.-4] mol/L) in the PG[F.sub.2][alpha]-precontracted endothelium-denuded rings from rats exposed to 40 ppm NO during the 10-day recovery period.

Reagents

The following drugs were used: acetylcholine hydrochloride, SNP, indomethacin, papaverine hydrochloride (Nacalai Tesque; Kyoto, Japan); PG[F.sub.2][alpha] (Ono Pharmaceutical; Osaka, Japan); and 8-bromo-cGMP (Sigma, St. Louis, MO). The concentrations of these drugs are expressed as the final molar concentrations in the organ bath. 8-Bromo-cGMP was stored at -20[degrees]C and diluted with distilled water just before each experiment. NO was obtained from Sumitomo Seika (Chiba, Japan).

Data Analysis

Results are expressed as means [+ or -] SE. Differences between two groups were determined by the unpaired Student t test. When means of more than two groups were compared, one-way analysis of variance was used. If a significant difference was found, a Fisher exact test was used to identify which groups were different; p < 0.05 was considered significant.

RESULTS

RVH

The RV/(LV+S) significantly increased in CH control rats with a recovery period of 3 days after CH compared with normal control rats, indicating the occurrence of RVH (Fig 1). The increase in RV/ (LV+S) in CH control rats was still observed after 10 days of recovery. NO inhalation during the recovery period did not affect the RV/(LV+S) in either normal or CH rats.

[FIGURE 1 OMITTED]

Response to Acetylcholine

< 10 ppm NO: The relaxation to acetylcholine was significantly reduced in IPA rings from CH control rats compared with those of normal control rats after both 3 days and 10 days of recovery (Fig 2). The relaxation was similar between rings from rats with and without 10 ppm NO inhalation irrespective of the recovery period. Similar results were obtained in the EPA (data not shown).

[FIGURE 2 OMITTED]

< 40 ppm NO: In both the EPA (data not shown) and the IPA, the relaxation was also similar between rings from rats with and without 40 ppm NO inhalation irrespective of the recovery period.

Response to SNP

< 10 ppm NO: The relaxation to SNP was markedly reduced in IPA rings from CH control rats compared with those of normal control rats (Fig 3). The relaxation was generally similar between rings from rats with and without 10 ppm NO inhalation irrespective of recovery period. Similar results were obtained in the EPA (data not shown).

[FIGURE 3 OMITTED]

< 40 ppm NO: In both normal and CH EPA rings, the relaxation was also similar between rings from rats with and without 40 ppm NO inhalation irrespective of the recovery period (data not shown). In CH IPA rings, the relaxation was again similar between rings from rats with and without 40 ppm NO inhalation irrespective of recovery period. But in normal IPA, 40 ppm NO inhalation for 10 days markedly augmented the relaxation in response to SNP at [10.sup.-9] mol/L and [10.sup.-8] mol/L.

Response to SNP and 8-Bromo-cGMP in Endothelium-Denuded 1PA Rings

SNP: In contrast to endothelium-intaet rings, SNP ([10.sup.-9] to [10.sup.-5] mol/L) induced similar relaxation in endothelium-denuded IPA rings from normal control and CH control rats in IPA (Fig 4). Continuous 10-day 40 ppm NO inhalation did not affect the relaxation caused by SNP in endothelinm-denuded IPA rings from CH rats. But in endothelinm denuded tings from normal rats, 10-day 40 ppm NO inhalation augmented relaxation to SNP at all concentrations examined.

[FIGURE 4 OMITTED]

8-Bromo-cGMP: 8-Bromo-cGMP induced similar relaxation in endothelinm-denuded tings from norreal control and CH control rats in IPA. Continuous 40 ppm NO inhalation for 10 days did not affect the relaxation caused by 8-bromo-eGMP in endothelium-denuded IPA tings from CH rats. But in IPA from normal rats, 40 ppm NO inhalation for 10 days augmented the relaxation response to 8-bromo-eGMP at all concentrations examined except [10.sup.-6] mol/L.

Urine N[O.sub.3] Concentration

The average value of the concentration in urine samples was significantly higher in the NO group (29.54 [+ or -] 5.59 mmol/L, n = 6) than in the air group (0.56 [+ or -] 0.05 mmol/L, n = 4; p < 0.05).

DISCUSSION

Prolonged NO inhalation did not affect the recovery process of acetylcholine- or SNP-induced relaxation in the CH hypertensive PA. In normal IPA, the relaxation in response to SNP was partially increased by long-term (10 days) inhalation of 40 ppm NO, whereas relaxation in response to acetylcholine remained unchanged. The relaxations to SNP and 8-bromo-cGMP were also augmented in endothelium-denuded normal IPA after 10 days of 40 ppm NO inhalation.

Ring studies have inherent limitations when ring preparation is made. It is difficult for us to work with ring studies in resistance PAs of rats. Although resistance vessels are important in hypoxic changes, conduit PAs might also play an important role because the changes of lumen size in conduit PAs affect blood flow to peripheral vessels. We previously described that functional alteration occurred in conduit PAs, (14) as well as resistance PAs during and after CH. In this study, we used EPA and IPA because IPA is directly exposed to inhaled NO, but not EPA.

A previous study (18) in our laboratory showed that continuous NO inhalation had no effect on regression of pulmonary vascular structural changes and RVH in recovery from chronic hypoxic PH. The present results confirmed that prolonged NO inhalation showed no effect on the regression of RVH. The purpose of the present study was to identify the effects of prolonged NO inhalation on the functional recovery of the PH vessel (ie, recovery of impaired NO-mediated relaxation caused by CH exposure).

Prolonged NO inhalation did not affect the endothelium-dependent relaxation responses to acetylcholine in normal or hypertensive conduit PAs. However, in isolated rat lung from normal (21) and CH PH, (19) middle- and long-term exposure to NO inhalation induced impairment of endothelium-dependent vasorelaxation. However, these studies did not find endothelial NO synthase (NOS) messenger RNA level alteration. Pulmonary resistance arteries had been highly exposed to inhaled NO (22) compared with conduit PAs. Isolated lung model reflected these pulmonary resistance arteries. Differences in the preparation used might explain the difference among results.

In clinical reports, (6,23) a significant rebound increase in pulmonary vascular resistance above the initial precondition level has been demonstrated after discontinuation of short-term NO inhalation. This phenomenon might be associated with impaired endothelial NOS, which maintains basal pulmonary vascular tone. Prolonged inhaled NO is used in patients with primary PH (70 and pulmonary fibrosis (20) Although elinical application of the present study is limited, prolonged NO inhalation does not decrease NO-dependent relaxation, at least in PAs of CH PH.

SNP-induced relaxation was partially augmented by long-term (10 days) inhalation of 40 ppm NO in both endothelium-intaet and endothelium-denuded normal IPAs. Continuous NO inhalation transiently suppressed total NOS activity in vivo (24) and in vitro, (11,12) Decreased NOS activity might partially cause impaired endogenous NO synthesis, which determines basal vascular tone. Dinerman et al (25) observed nitroglycerin (NO donor)-induced vasorelaxation was increased in endothelium-denuded segments of rat thoracic aorta compared with endothelium-intact segments. These results suggested that basal endogenous NO produced and/or released from endothelial ceils might modulate relaxing effect of exogenous NO in competition with each other. Nakagawa et al (26) also described greater accumulation of cGMP induced by nitroglycerin-mediated vasorelaxation in de-endothelialized canine coronary arteries compared with endothelium-intaet segments. It is possible that decreased basal endogenous NO induced by continuous NO inhalation or endothelium denudation might reduce competitive interaction with exogenous NO and cause augmented SNP-mediated vasorelaxation.

Prolonged NO inhalation might have affected the normal endogenous NO-cGMP relaxation pathway in intact pulmonary vaseulature, but not in an already impaired pathway in hypertensive PAs. To induce functional changes in the normal PA by prolonged NO inhalation, a high NO concentration and a long-term exposure period might be necessary, because only 40 ppm (high concentration) for 10-day (long period) exposure had an effect on intact PAs in this study.

To further examine the mechanism of the slightly augmented endothelium-independent relaxation, we tested relaxation in response to SNP and 8-bromo-cGMP in endothelium-denuded normal IPA rings. As the relaxation to both SNP and 8-bromo-cGMP was augmented in endothelium-denuded normal IPA rings exposed to 40 ppm NO inhalation for 10 days, this might suggest that smooth-muscle reactivity to eGMP is increased by prolonged NO inhalation in normal IPA.

The impairment of SNP-induced relaxation was completely restored after denudation of endothelium in the hypertensive IPA, suggesting smooth-muscle cell reactivity to NO is not altered by CH. Prolonged NO inhalation had no effect on this restoration by endothelium denudation. Several experiments (16,27) demonstrated that impaired eGMPinduced vasorelaxation might be related to increased phosphodiesterase 5 (PD[E.sub.5]) activity, cGMP binds to the noncatalytic binding site of PD[E.sub.5] and induces phosphorylation. NO and nitrovasodilators act as relaxants by direct activation of guanylyl cyclase. It is necessary to determine whether endotheliumderived NO indirectly activates PD[E.sub.5] in smooth muscle cells in CH PH. In summary, combined with previous study, (18) prolonged NO inhalation does not affect the regression of already developed functional and structural changes induced by CH in the pulmonary vasculature.

* From the Departments of Physiology (Dr. J. Maruyama) and Anesthesiology (Drs. Jiang and K. Maruyama), Mie University School of Medicine, Mie; and Pathophysiology Research Laboratory (Drs. Takata and Miyasaka), National Children's Medical Research Center, Tokyo, Japan.

This work was supported in part by Grants-In-Aid for Scientific Research 07771228 and 08770045 from the Japanese Ministry of Education, Science and Culture, and Grants for Pediatric Research 5C-02 and 8C-02 from the Ministry of Health and Welfare.

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Manuscript received February 4, 2004; revision accepted July 13, 2004.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: permissions@ehestnet.org).

Correspondence to: Junko Maruyama, MD, Department of Physiology, Mie University School of Medicine, 2-174, Edobashi, Tsu, Mie 514-8507, Japan; e-mail: j-maru@doc.medic.mie-u.ac.jp

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