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J Appl Physiol 89: 1971-1978, 2000;
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Vol. 89, Issue 5, 1971-1978, November 2000

Airway function after cyclooxygenase inhibition during hyperpnea-induced bronchoconstriction in guinea pigs

O. E. Suman1, J. D. Morrow2, K. A. O'Malley1, and K. C. Beck1

1 Thoracic Diseases Research Unit, Division of Pulmonary and Critical Care Medicine, Mayo Clinic and Foundation, Rochester, Minnesota 55905; and 2 Division of Clinical Pharmacology, Department of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Airway function deteriorates significantly on cessation of exercise or isocapnic hyperventilation challenges but is largely preserved during the challenge in humans and guinea pigs. PGE2, an endogenous bronchodilator, might be responsible for the preservation of lung function during hyperventilation (HV). We hypothesized that PGE2 might have a protective effect during HV, partially explaining the minimal changes in respiratory system resistance (Rrs) usually seen during HV in humans and guinea pigs. Therefore, changes in Rrs were measured during and after HV in anesthetized, mechanically ventilated guinea pigs treated with flurbiprofen (FBN) or placebo. With HV, there was an initial bronchodilation that was unaffected by FBN. Rrs then increased with time during HV, an effect that was blocked by FBN. After HV, Rrs increased further in all groups, but the increase in Rrs was less in the FBN-treated groups. FBN treatment reduced the PGE2 concentration slightly in lung lavage fluid compared with placebo. We found no enhancement or refractoriness of the Rrs response to repeat bouts of HV and no effect of FBN treatment on the response of Rrs to repeat HV. These results suggest that a constrictor PG is released during and possibly after HV and that the post-HV increase in Rrs is the sum of effects of the PG released during HV and a second constrictor mechanism operating after HV. We found no evidence for bronchodilator PG during or after HV in the guinea pig.

exercise-induced asthma; isocapnic hyperventilation; prostaglandins; arachidonic acid; respiratory system resistance


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AIRWAY FUNCTION DURING EXERCISE or isocapnic hyperventilation (IH) is preserved in asthmatic subjects (4, 7, 45, 46). On cessation of the hyperpnea, airway function deteriorates significantly. The physiological mechanism for this apparent protective effect on airway function during hyperpnea is unknown. Guinea pigs show a time-dependent response in lung function during and after hyperventilation (HV) that is similar to the response of asthmatic humans (39, 41), and guinea pigs have been utilized as a model of airway hyperresponsiveness (9, 23, 40, 41). A leading candidate as the mediator involved in preserving airway function during hyperpnea is PGE2. PGE2 is a known bronchodilator produced in the lungs and bronchi (38) of humans (19, 33, 34, 38) and guinea pigs (6, 10). Tallet and colleagues (47) reported the release of PG from stretched canine airway smooth muscle in vitro and demonstrated inhibition of smooth muscle tone by PGE2. PGE2 and its effect on lung function during IH (in vivo) have not been studied. In this study we used flurbiprofen (FBN), a potent cyclooxygenase inhibitor, to investigate the role of PG in affecting airway function during and after IH in guinea pigs. We hypothesized that PGE2 might have a protective effect during HV, partially explaining the minimal changes in respiratory system resistance (Rrs) usually seen during HV in humans and guinea pigs (4, 7, 39, 41, 45, 46). To this end, we measured PGE2 concentration in lung lavage to determine whether it was present after the IH and evaluated Rrs changes.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Male Hartley guinea pigs (n = 96, 633.4 ± 66.2 g body wt) were randomly assigned to one of three ventilation groups defined by ventilator settings and one of two drug groups defined by the type of drug administered (Table 1).

                              
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  		Table 1.   Guinea pig randomization scheme

Anesthesia and intubation. With the use of a randomization schedule, each guinea pig received orally 0.1 ml of ethanol or 10 mg/ml of FBN (2.0 mg/kg) dissolved in 0.1 ml of ethanol, defining the placebo and FBN groups, respectively. Then each animal was anesthetized with a mixture of ketamine and xylazine (100 mg/ml ketamine added to 1 ml of xylazine, 1 ml/kg body wt), injected into a muscle of the lower extremity. Once anesthetized, animals were intubated through a tracheotomy and the lungs were ventilated at a volume of 5.5 ml/kg with the use of a small animal ventilator (Harvard Apparatus, Millis, MA) attached to the animal via 2-in. silicone rubber tubing (2.0 mm ID) with a Y piece at the end. A catheter was inserted into the jugular vein for administration of the paralytic agent vecuronium. The animal was placed inside a small animal body plethysmograph. After respiratory system mechanics were monitored for 25-30 min, 0.1 ml of vecuronium (1.0 mg/ml iv) was given to prevent breathing efforts from affecting subsequent Rrs measurement.

Pulmonary mechanics. Pressure at the mouth was measured with a calibrated pressure transducer (range ±200 cmH2O, Celesco Transducer Products, Canoga Park, CA) connected to a side tap of the endotracheal tube. Pressure in the small animal plethysmograph was measured using a ±2-cmH2O pressure transducer (Celesco Transducer Products). The latter pressure was proportional to flow across a screen pneumotachograph mounted in the rear wall of the plethysmograph. The flow signal was integrated digitally to produce a volume signal, which in turn was corrected for pressure in the plethysmograph by digitally subtracting a signal proportional to the pressure. The proportionality constant was adjusted to bring integrated box volume and integrated mouth volume signals into phase when the empty plethysmograph was pumped with a small animal ventilator. The box flow signal was calibrated on each study day using a 5-ml gas-filled syringe to produce a known volume signal. The two pressure signals were fed into a personal computer-based data acquisition system at rate of 180 s-1.

Rrs was measured on a breath-by-breath basis. Respiratory system compliance (Crs,dyn) was obtained from Delta VL/Delta Pm, where Delta VL indicates the change in volume from the beginning to the end of inspiration and Delta Pm is the change in mouth pressure over the same interval. Rrs was then obtained from the slope by linear regression of the plot of flow vs. [Pm - (Delta VL/Crs,dyn)], which is equivalent to pressure change divided by change in flow (range of flow ±20 ml/s) (12). Data for at least five breaths were averaged at each time point of measurement.

Hyperpneic protocol. Pre-HV measurements of tidal volume, mixed expired CO2, respiratory rate, and Rrs were made every 2 min for 10 min. At each recording time, data for 10 breaths were recorded and then a maximal inspiration was delivered via a syringe (20-25 cm of maximal airway pressure). Rrs was measured by averaging data from breaths before the deep inspiration. After 10 min, individual guinea pigs were randomly assigned to one of three ventilation strategies: a normal ventilation (NV) control group and two HV groups (Table 1). In the NV group, the ventilator was kept at 40 breaths/min and a constant tidal volume was maintained throughout the experiment. In the increased frequency group (HV150), the ventilator rate was increased to 150 breaths/min while tidal volume was maintained at resting levels during the 10 min of HV. In the second HV group (HV100), the respiratory rate was increased to 100 breaths/min and the tidal volume was increased by 1.5-2.0 ml above pre-HV values for 10 min of HV. To maintain eucapnea, 5% CO2 was mixed with compressed, dry room air in the inspired port of the ventilator. During the 10 min of hyperpnea, Rrs was recorded immediately at the onset of HV and then every 2 min without the deep inspirations. After the HV challenge, the ventilator was returned to prechallenge settings (i.e., 40 breaths/min and 5.5 ml/kg) for 4 min. Rrs was recorded every 2 min, again without deep inspirations.

Lung lavage collection. At 4 min after the hyperpnea challenge, each animal underwent lung lavage with 10 ml of warmed sterile saline (37°C). Saline was injected and withdrawn five times through the endotracheal tube. The recovered fluid volume was recorded, collected into a polypropylene tube, and centrifuged for 20 min at 2,000 g and 4°C under nitrogen. The cell-free supernatant was then aliquoted (400-1,000 µl) into propylene tubes that were closed under nitrogen and stored at -70°C for later ELISA of PGE2.

PGE2 release was assessed using the ELISA (Cayman Chemical, Ann Arbor, MI) of 50 µl of cell-free lung lavage supernatant. All samples were processed and stored in identical fashion. Aliquots from a number of samples were assayed more than once to check the reproducibility of the assay and for possible decay of PGE2 in the samples in cold storage.

Validation of the ELISA. PGE2 levels obtained with the ELISA method were validated by directly comparing results obtained by ELISA with gas chromatography followed by mass spectrometry. In 11 samples, the lung lavage was split into 2 aliquots immediately after lavage. In one of the aliquots, a known amount of PGE2 was added to test the measured recovery using the two PGE2 recovery analysis methods.

Repeat HV. Two additional groups of guinea pigs were utilized to evaluate the role of prostanoids after repeated bouts of HV and were randomly assigned to placebo (n = 12) or FBN (n = 12) drug groups.

The anesthesia and intubation procedures were identical to those described above. After pre-HV measurements were recorded, the ventilator setting was increased in all guinea pigs to 1.5-2.0 ml above the resting tidal volume and a breathing frequency of 100 breaths/min. In contrast to the first part of our experiment, lung lavage was not collected. After the initial HV period, guinea pigs were monitored for 12 min with the ventilator set at 40 breaths/min and a resting tidal volume. After 12 min, the ventilator settings were increased to 100 breaths/min and a tidal volume of 1.5-2.0 ml above resting levels for a repeat HV period of 10 additional min. During the 10 min of HV, Rrs was recorded immediately at the onset of HV, then every 2 min without the deep inspirations. At the end of the second ventilation period, the ventilator settings were returned to resting levels for 6 min. Rrs was measured 2 and 4 min after the second bout of HV.

Thoracic cage dimensions. Because changes in lung volume during HV might have influenced Rrs, we indirectly documented changes in lung volume during HV. To this end, we used mercury strain gauges to determine changes in the outer dimension of the lower rib cage in six animals: three HV150 and three HV100 animals.

The six animals were anesthetized and paralyzed, and a tracheotomy was performed as in the main study. The animals were not given further drug treatments. A mercury strain gauge consisting of silicone rubber tubing containing mercury (16-18 cm long, D. E. Hokanson, Bellevue, WA) was wrapped around the lower thoracic cage of each guinea pig ~5-10 min before the start of HV. Mercury strain gauges produce a voltage output that is proportional to changes in length of the mercury-containing silicone rubber tubing, which in this case indicated changes in chest wall circumference. Strain gauges were calibrated by taking a voltage reading at end expiration and after full inflation to mouth pressure of 25-30 cmH2O (i.e., after inspiratory capacity volume was injected with a 10-ml syringe). All subsequent strain gauge measurements were referenced to these values, and strain gauge values are reported as a fraction of the full inspiratory capacity voltage excursion. After installation of the strain gauges, animals were placed in the body plethysmograph, monitored for 5-10 min, and then subjected to 10 min of HV as in the main study.

Data analysis. The changes in pulmonary function during the early stages of HV were assessed by comparing Rrs at 2 min with the pre-HV value. The change in Rrs during all stages of HV was documented by the linear regression slope of Rrs against time for each animal. The fractional bronchoconstrictor response after HV (PP) was determined by comparing the largest Rrs measured after HV with that before HV [(largest post - pre)/pre]. The immediate bronchoconstrictor fractional response (IP) was similarly documented by comparing the highest post-HV Rrs value with the last Rrs measurement during HV (10 min) [(largest post - 10 min)/10 min].

Statistical analysis. PGE2 levels and Rrs values are means ± SE. Because there was considerable variability in Rrs and PGE2 measurements and the variability was not constant in the various groups, logarithmic transformations were performed to meet the requirement of homoscedascity for ANOVA. Repeated- and nonrepeated-measures ANOVA were used to test for the effects of ventilation strategy and drug treatment on Rrs across time. Tukey's t-tests were conducted to compare ventilation and drug groups. To test for changes in Rrs during HV, the slope of Rrs vs. time was submitted to ANOVA and subsequent Tukey's t-tests to test effects of drug and ventilation strategy. P < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Does Rrs change during HV? Figure 1A reflects no significant change (P > 0.05) in Rrs across time in normally ventilated animals (40 breaths/min, 5.5 ml/kg) in the two drug groups. In addition, there was no effect of FBN treatment on Rrs at any time point.


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  		Fig. 1.   Fractional changes from baseline in respiratory system resistance (Rrs) for all sessions according to drug treatment. A: changes in Rrs from baseline levels throughout the session during which the ventilator was kept at 40 breaths/min. B: changes in Rrs from baseline when the ventilator was increased to 150 breaths/min during the isocapnic hyperventilation portion of the session. C: same as B, except the ventilator was increased to 100 breaths/min and the tidal volume (VT) was increased to 1.5-2.0 times the baseline VT. Values are means ± SE; n = 16 for each of the 6 groups. *P < 0.05 for the slope of Rrs response between placebo- and flurbiprofen (FBN)-treated groups. Gray shaded area represents period of hyperventilation (HV). f, Breathing frequency.

However, with HV (Fig. 1, B and C), there was an initial bronchodilation in the two HV groups that was not affected by FBN. The 2- to 10-min slopes of Rrs vs. time for the placebo HV150 and HV100 groups were significantly steeper than the slopes for NV groups and corresponding FBN-treated groups, indicating a steeper rise in Rrs with HV caused by a PG-dependent bronchoconstrictor mechanism during HV.

Does Rrs change after HV? Rrs showed a marked increase up to 4 min after HV for all HV groups (Fig. 1). Figure 2 shows the PP (prehyperpnea vs. highest posthyperpnea Rrs) and the IP (10 min vs. highest posthyperpnea Rrs) changes in Rrs after HV in all animals. ANOVA indicated differences in the PP response between the two drug groups and between the HV groups (P < 0.05). The PP response in the two HV groups was reduced by FBN. In contrast, the IP Rrs response was not affected by FBN.


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  		Fig. 2.   Calculated percent changes in Rrs after HV. triangle , Percent changes in Rrs of the largest post-HV value relative to pre-HV baseline (PP); open circle , immediate percent changes in Rrs of the largest post-HV value relative to the last HV time point (10 min; IP). Each point represents data from 1 animal. Data are grouped by ventilation strategy and by drug treatment. ANOVA indicated a significant drug and ventilation effect on the PP response but no drug effect on the IP response. *P < 0.05 between placebo and FBN comparison. Horizontal bars represent means for a given group.

Are PGE2 concentrations in lung lavage elevated after HV? Figure 3 shows the average concentrations of immunoreactive PGE2 in lung lavage for all groups. The transformed data [ln(PGE2)] are shown in Fig. 3, bottom. Despite the variability and considerable overlap among groups, ANOVA demonstrated that treatment with FBN lowered the mean PGE2 concentration slightly compared with placebo. However, there was not a significant difference in PGE2 among the ventilation groups (including NV). This suggests that PGE2 is being released but that its rate of release is not affected by the mode of ventilation.


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  		Fig. 3.   Top: absolute levels of PGE2 measured in the lung lavage (LL) of animals. Lung lavage samples were collected after the 4-min post-HV time period. Each point represents data from 1 animal. Data are grouped by ventilation strategy and by drug treatment. Horizontal bars represent means for a given group. Bottom: logarithmic (ln) representation of the absolute PGE2 levels. Note the degree of variability in PGE2 concentration and the considerable overlap among the various groups. *P < 0.05 between placebo and FBN group comparison.

We compared ELISA with gas chromatography followed by mass spectrometry for PG-spiked and nonspiked samples. This comparison yielded a correlation coefficient of 0.82. In addition, repeat analysis of split aliquots using ELISA showed no significant decay in PGE2 concentration with time in storage (P > 0.05). From these results, we concluded that the ELISA was able to detect changes in PGE2 with sufficient reliability and an acceptable validity.

Could prostanoid-dependent effects be manifested during repeat HV periods? Two groups of animals were studied to determine the effects of FBN treatment on response to repeated HV (Fig. 4). After the initial HV and repeat HV, the PP response in Rrs was reduced by FBN treatment, as in the main study. However, the comparison between initial PP response and repeat PP response in Rrs showed no significant drug effect, indicating no PG-dependent alteration in responsiveness on repeat HV. The IP response, unlike the main study group, was larger in the placebo-treated group than in the FBN-treated group for the initial HV and repeat HV. However, paired comparison of repeat vs. initial IP response again showed no significant drug effect. The slope in Rrs during HV of the initial HV period was steeper in the placebo- than in the FBN-treated group, although the slopes in the repeat HV period were not different. This suggested that the PG dependence of the increase in Rrs during HV became less on repeat HV, although a paired comparison of slopes between initial and repeat HV just missed statistical significance (P > 0.05). In summary, paired testing showed no significant effect of FBN treatment on the PP or IP response to repeat HV. The prostanoid-dependent rise in Rrs during HV might be blunted by repeat HV; however, a larger group of animals would be needed to prove this.


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  		Fig. 4.   Changes in Rrs during and after repeat periods of HV. Both groups of guinea pigs (n = 12 for placebo and FBN) were initially ventilated with 100 breaths/min and increased VT for 10 min. After the initial bout of hyperpnea, both groups were allowed to recover for 12 min and were subsequently ventilated a second time with 100 breaths/min and increased VT for 10 min. Values are means ± SE. *Statistical significance between placebo and FBN groups, P < 0.05. Paired comparison of PP, IP, and slope differences between initial HV and repeat HV periods showed no significant drug effect. NS, no significant difference in the slope of Rrs between drug groups during HV of the repeat bout of HV. Gray shaded area represents the period of HV.

Lung volume changes during HV. The changes in strain in the HV150 group are shown in Fig. 5. Average tidal strain change was constant during HV, indicating adequate control of tidal volume during HV (Fig. 5, top). Figure 5, bottom, shows changes in end-expiratory, end-inspiratory, and mean strain. At onset of HV there was a slight increase in end-expiratory (3%), end-inspiratory (6.5%), and mean (4.8%) strain. There was a slow downward drift in the chest wall circumference measurements during HV. When HV stopped and frequency returned to 40 breaths/min, there was a drop in mean strain of 8.9% of the inspiratory capacity excursion.


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  		Fig. 5.   Changes (means ± SD) in strain gauge output (thoracic circumference) in 3 guinea pigs hyperventilated with 150 breaths/min and no change in VT. EE and EI, end-expiratory and end-inspiratory, respectively. Mean strain is mean of EE strain and EI strain. Top: EE-EI strain differences. Hyperventilation started at time 0 and continued to minute 10. Gray shaded area represents the period of HV.

The change in strain gauge output for the HV100 group is shown in Fig. 6. The tidal excursion of the strain gauge increased as expected with the increased tidal volume in this group. Similarly, end-expiratory strain increased by about the same amount as in the HV150 group (~5% of inspiratory capacity). Again, similar to the HV150 group, there was a slow drift downward in the strain signal and a 7% drop in strain at the end of HV. The increases in end-inspiratory (35.5%) and mean (20%) circumference were larger than in the HV150 group.


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  		Fig. 6.   Changes (means ± SD) in strain gauge output (thoracic circumference) in 3 guinea pigs hyperventilated with 100 breaths/min and 50% increase in VT. Top: EE-EI strain differences. Hyperventilation started at time 0 and continued to minute 10. Gray shaded area represents the period of HV.

Volume changes were roughly estimated assuming isotropic expansion of lungs and chest wall in all dimensions and V proportional to  C3 (where V is volume and C is circumference). These calculations suggested increases in mean lung volume of 15 and 74% in the HV150 and HV100 groups, respectively, at the start of HV. The increases in end-expiratory volumes were similar in both groups (10-15%).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We hypothesized that PGE2 might have a protective effect during HV, partially explaining the minimal changes in Rrs usually seen during HV in humans and guinea pigs (4, 7, 9, 39, 41, 45, 46). Both forms of HV (increased rate only and increased rate and tidal volume) induced an initial bronchodilation, which slowly reversed as the hyperpnea progressed. However, the upward slope in Rrs seen during HV was attenuated, and the fractional increase in Rrs after HV was significantly reduced by FBN treatment. These results suggest that a constrictor PG is released slowly during HV and that the post-HV increase in Rrs is the sum of the effects of PG released during HV and a second constrictor mechanism operating after HV. To further substantiate this, we found no increase in concentrations of PGE2 in lung lavage fluid after HV. In contrast to humans, in whom there are reports of refractoriness in exercise and HV, we found neither refractoriness nor enhancement of the Rrs response to repeat bouts of HV in guinea pigs.

Limitations of the study. There were concerns about the effects of ketamine and vecuronium on Rrs. Because all groups received ketamine and vecuronium, any effect specifically caused only by ketamine or vecuronium would have been manifested in all groups. Although ketamine is an airway smooth muscle relaxant, it did not prevent the post-HV response or the progressive bronchoconstrictor response during HV. We cannot rule out that ketamine or vecuronium blunted or enhanced the progressive increase in Rrs during HV or the post-HV bronchoconstrictor response. However, in placebo and FBN unventilated groups, Rrs remained unchanged after administration of ketamine and vecuronium. In addition, administration of ketamine and vecuronium did not maximally relax airway smooth muscle, because at the onset of HV all groups decreased Rrs initially.

It has been suggested that vecuronium (37) has physiological effects in human airways by inhibiting cholinesterase and muscarinic (M3) presynaptic receptors. Inhibiting presynaptic receptors could have amplified the bronchoconstrictor response measured in our study if the response was vagally mediated. However, both groups were given an identical amount of vecuronium, which could therefore not have been the sole cause of our diverging degree of airway obstruction in our animals. The post-HV increase in Rrs is not vagally mediated in guinea pigs (41).

Ideally, we would have used a specific PGE2-inhibiting agent to demonstrate the role of PGE2 in this animal model of HV-induced asthma. To our knowledge, specific PGE2 receptor blockers or synthesis inhibitors are not available. In this study we were seeking evidence for a bronchodilator prostanoid such as PGE2. However, by blocking cyclooxygenase synthesis, bronchoconstrictor and bronchodilator prostanoids will be inhibited. Our results support dominance of constrictor prostanoids, because we found little increase in the PGE2 concentrations with HV, and we found cyclooxygenase inhibition to have a net bronchorelaxant effect during and after HV. PGs have also been implicated as a cause of refractoriness to repeat exercise bouts in humans (31). However, the dilator effect of PGs associated with refractoriness has been observed only during exercise, not during HV (32).

Our results indicate no refractoriness or enhancement of the bronchoconstrictor response to repeat HV challenges in guinea pigs. However, our results could be interpreted as two different constrictor PGs being released (one during and one after HV), with the PG during HV becoming depleted and not being involved in the second HV period. Although the guinea pig has been used as a model for HV-induced asthma and as a model of hyperreactivity (9, 23, 24, 35, 39, 41), caution is still warranted in extrapolating results from animal studies to humans. Ray and colleagues (39, 41) have shown that the time course and degree of bronchoconstrictor response are similar between humans and guinea pigs. In addition, the pulmonary response during IH with dry gas is also similar between guinea pigs and humans. Humans demonstrate a lesser response to repeat bouts of exercise and HV. We do not know whether guinea pigs would show such refractoriness to repeat exercise, but their response to repeat HV does not suggest refractoriness (in contrast to humans). It is apparent that the post-HV bronchoconstriction is affected primarily by tachykinin in guinea pigs (40) in addition to leukotrienes but by histamine, leukotrienes, PGs, and vagal nerve stimulation in humans (11, 20, 21, 27, 29-31, 45). Despite these differences, it is useful to explore mechanisms for bronchoconstrictor response to HV in animals. The mediators or mechanisms responsible for the bronchodilation or preservation of lung function observed during HV in humans and guinea pigs are unknown and could share common mechanisms.

Another limitation is the effect of ethanol (the vehicle for FBN) on the preservation of lung function. A previous study demonstrated a bronchodilator effect of ethanol (1, 2). However, we attempted to keep the dose of ethanol low, and we compared FBN in ethanol vehicle with the ethanol vehicle alone so that any effect of ethanol would have operated in both groups.

We used lung lavage fluid to document changes in PGE2 production with HV and found little effect of HV on PGE2 production. This negative result is not as conclusive as a positive result. One could still hypothesize that PGE2 concentrations in the tissues were elevated by HV and that the dilution or inaccessibility of interstitial fluid assessed via lavage caused an underestimation of PGE2 tissue levels. However, results of FBN treatment indicate that there is no net bronchodilating PG produced by HV in guinea pigs.

Mechanisms. The mechanisms operating to maintain airway function during HV are unknown. Substances or conditions that might have a bronchodilator influence include PGE2 (15, 19, 38, 42), nitric oxide (5, 22, 25, 48), lung stretch (13, 14), and calcitonin gene-related peptide (36). Other naturally occurring bronchodilators include S-nitrosothiol (18), circulating catecholamines (3), and other PGs (PGE1 and PGI2) (15, 44).

PGE2. Our results showed no net bronchoconstrictor effect of cyclooxygenase inhibition by FBN during or after HV in guinea pigs. Although PGE2 has been shown to relax isolated bronchial smooth muscle in guinea pigs (42) and to have protective effects against exercise- or HV-induced bronchoconstriction in asthmatic patients (33, 34, 38), we could not substantiate a role for exogenously released dilator PG in our experiments. Despite the evidence for a possible role for PGE2 as a bronchodilator mediator, our findings are in agreement with those of Savla and colleagues (43), who found a decrease in PGE2 production with cyclic stretching of cat and human airway epithelial cells in vitro.

Nitric oxide. Nitric oxide also reduces airway tone in guinea pigs and asthmatic patients (22, 25). Nitric oxide is thought to be the neurotransmitter of the nonadrenergic, noncholinergic nerves, which could be activated during exercise (5, 28). Nitric oxide induced bronchodilation and attenuated the smooth muscle response to methacholine in isolated guinea pig trachea (28). In another study, Yoshihara and colleagues (48) reported inhibition of bronchoconstriction induced by cold air inhalation by endogenous nitric oxide. To our knowledge, the role of nitric oxide as a bronchodilator during exercise or hyperpnea is unknown.

Increased lung volume (stretching). There are three possible mechanical effects of HV that could cause a reduction in Rrs: increases in mean lung volume, absolute tidal volume excursion, and an increase in respiratory rate. To detect change in mean lung volume, we measured changes in chest wall circumference in six animals (Figs. 5 and 6). In the HV150 group (no change in tidal volume), mean lung volume changed only minimally (~15%), but the HV100 group experienced larger changes in mean lung volume (~74%) and a higher end-inspiratory volume. Despite this difference in lung volume changes, the initial fall in Rrs at onset of HV was the same in both groups, suggesting little role for lung volume changes per se. Increased rate could also have a bronchodilator effect: in other species, pulmonary resistance drops with increasing frequency, probably because of a decreased effective tissue resistance (8, 26). Our results suggest that in guinea pigs there is a drop in Rrs with an increase in frequency between 40 and 100 breaths/min but little change between 100 and 150 breaths/min. However, our studies were not designed to examine the frequency dependence of Rrs in detail.

We found that Rrs during HV showed an initial decrease followed by a steady increase in placebo-treated animals. The slow rise in Rrs during HV was completely blocked by FBN. However, the IP response in Rrs was not affected by FBN, suggesting that the IP response was caused by non-PG mechanisms such as tachykinins or leukotriene release in this species (43). The sum of these two responses should equal the PP response. Thus the PP response was partly blunted by FBN (Figs. 1, B and C, and 2, top), consistent with a model of slow release of PG during HV but other (predominantly nonprostanoid) mechanisms operating after HV. From this study, the following model for response to HV emerges. During HV, there is an initial fall in pulmonary resistance caused predominantly by an increase in respiratory rate, although other active non-prostanoid bronchodilator mechanisms cannot be ruled out. During continued HV, the rate of release of bronchoconstrictor PG slowly increases, perhaps as a result of airway drying, cooling, or stretch, causing a slow rise in Rrs. Full bronchoconstriction does not develop because of effects of airway cooling (15-17) or mechanical effect of tidal stretches on cross-bridge function in airway smooth muscle (8, 13, 14). When HV stops, full expression of the bronchoconstrictor effect by release of additional non-prostanoid constrictor mediators or mechanisms is manifested (Fig. 1, B and C). During a second HV period, there is again an initial bronchodilation but no rise in Rrs during HV (placebo or FBN), suggesting possible inactivation of HV-induced PG release.

To summarize, we provide evidence for the slow release of a constrictor PG during HV in guinea pigs. On the other hand, we found no evidence for dilator PG involvement in the control of airway function during or immediately after HV, nor did we find evidence that PG involvement caused refractoriness to repeat HV in guinea pigs.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-52230.


    FOOTNOTES

Address for reprint requests and other correspondence: O. E. Suman, Medical Staff, Shriners Burn Institute; 815 Market St., Galveston, TX 77550 (E-mail: oesuman{at}utmb.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.

Received 28 October 1999; accepted in final form 19 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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J APPL PHYSIOL 89(5):1971-1978
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