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Am J Physiol Lung Cell Mol Physiol 294: L964-L973, 2008. First published March 7, 2008; doi:10.1152/ajplung.00401.2007
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Reduced spontaneous relaxation in immature guinea pig airway smooth muscle is associated with increased prostanoid release

Lu Wang,1 Valeria Pozzato,1 Graziella Turato,2 Aasakiran Madamanchi,1 Thomas M. Murphy,1 and Pasquale Chitano1

1Division of Pediatric Pulmonary Medicine, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina; and 2Divisione di Pneumologia, Dipartimento di Scienze Cardiologiche, Toraciche e Vascolari, Università degli Studi di Padova, Padova, Italy

Submitted 26 September 2007 ; accepted in final form 3 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Airway smooth muscle (ASM) from infant guinea pigs has less spontaneous relaxation during stimulation than ASM from adults. Inhibition of cyclooxygenase (COX), which catalyzes the production of prostanoids, increases this relaxation in infant ASM and abolishes age differences, thus suggesting that prostanoids reduce relaxation in infant ASM. In this study, we investigated whether leukotrienes are also involved in reducing spontaneous relaxation; whether the two COX isoforms, COX-1 and COX-2, differentially regulate spontaneous relaxation; and whether prostanoid release is developmentally regulated in guinea pig ASM. In different age groups, we measured relaxation during and after electrical stimulation in tracheal strips as well as prostanoid release from tracheal segments. Relaxation was studied in the absence and in the presence of a lipoxygenase inhibitor, a cysteinyl leukotriene receptor-1 antagonist, a COX-1 inhibitor, or a COX-2 inhibitor. We found that inhibition of lipoxygenase or cysteinyl leukotriene receptor-1 antagonism did not increase spontaneous relaxation at any age, thus excluding a role for leukotrienes in this phenomenon. Inhibition of COX-2, but not COX-1, promoted spontaneous relaxation. The basal release of prostanoids was more abundant in tissue from infant animals and decreased significantly with age. Thromboxane B2 was the most abundant metabolite released at all ages. Electrical stimulation and epithelium removal did not affect the age difference in prostanoid release. We conclude that increased basal prostanoid release contributes to the reduced spontaneous relaxation in immature guinea pig ASM compared with older animals. By regulating ASM relaxation, prostanoids may play a role in the airway hyperresponsiveness at a young age.

airway hyperresponsiveness; bronchospasm removal; cyclooxygenase; lipoxygenase; ontogenesis

AIRWAY HYPERRESPONSIVENESS is the exaggerated airway narrowing that occurs when airways are exposed to stimulants such as pharmacological agonists and nonspecific irritants. Hyperresponsiveness is consistently observed in response to stimuli that either directly activate receptors on airway smooth muscle (ASM) or indirectly contract ASM by causing the release of mediators (37). Common examples of direct stimuli are cholinergic agonists, histamine, and cysteinyl leukotrienes, whereas an indirect response may be caused by stimuli such as adenosine or hypotonic aerosols. While the exact contribution of structural and functional mechanisms leading to persistent airway hyperresponsiveness is still under investigation, an important and primary role has recently been shown to be played by the ASM. Isolated airway myocytes from transbronchial biopsies of asthmatic subjects revealed a greater shortening velocity and capacity than those from normal control volunteers (27), a feature that has evident relevance to the exaggerated airway narrowing in asthmatics. Airway hyperresponsiveness is not only a central feature of asthma and other obstructive airway diseases, but also characteristically occurs in healthy children and young animals (22, 35, 41). Notably, ASM function varies with maturation, expressing both a more pronounced contractile ability (9, 10, 49) and a reduced spontaneous relaxation (8) early during development. Both in disease and in immature individuals, some of the mechanisms causing airway hyperresponsiveness are likely inherent in the factors that regulate contraction and relaxation of ASM. Moreover, the altered function of immature ASM may contribute to a window of vulnerability that aggravates the impact of adverse events during early life and in turn may lead to a persistent asthmatic phenotype.

While the mechanisms of ASM contractile function have been widely studied, little is known about the regulation of ASM spontaneous relaxation (8). Apart from its ability to relax in response to pharmacological agents, we and others (8, 12, 15) have shown that healthy mature ASM has the intrinsic capability to spontaneously relax in the presence of stimulation. We defined this type of relaxation as spontaneous relaxation. We also defined parameters to describe the extent and rate of this relaxation to allow for comparison among various preparations. Furthermore, we postulated that a reduced extent of spontaneous relaxation during exposure to stimulants could contribute to airway responsiveness by maintaining bronchoconstriction and high airway resistance. Although it is currently unknown whether the intrinsic capacity to spontaneously relax is impaired in ASM from asthmatics, we have demonstrated a remarkable reduction of spontaneous relaxation in hyperresponsive-immature guinea pig trachealis (8). We showed that inhibition of cyclooxygenase (COX) with indomethacin restores ASM spontaneous relaxation in younger and hyperresponsive animals to resemble that of mature animals. This effect of indomethacin suggests that COX products, i.e., prostanoids, inhibit spontaneous relaxation and that the ontogenesis of guinea pig ASM spontaneous relaxation might originate from differences in the amount/profile of arachidonic acid metabolites in tracheal tissue.

COX exists in two isoforms: the constitutive COX-1 and the inducible COX-2. COX converts free arachidonic acid into prostaglandin H2, which in turn serves as substrate to different synthases, each catalyzing the production of one of the active prostanoids (47). Different prostanoids exert different direct effects on ASM (6, 30, 36), but they also affect ASM function indirectly through neural regulation (2, 16, 23, 48). It has been shown that prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2) are mainly relaxing agents, whereas prostaglandin F2{alpha} (PGF2{alpha}), prostaglandin D2 (PGD2), and thromboxane A2 (TXA2) are mainly contractile (6, 36). Besides being converted to prostanoids through the COX pathway, free arachidonic acid is utilized by the enzyme lipoxygenase to generate another family of compounds, leukotrienes (20). Leukotrienes also have direct effect on ASM by inducing a contractile response and increasing ASM responsiveness to other stimuli (39, 40, 52). They may therefore reduce the extent of ASM spontaneous relaxation, thus contributing to the ontogenetic differences we have previously reported in guinea pig trachealis. The generation of arachidonic acid metabolites may vary substantially in different tissues and play a role in childhood asthma (14, 42). It is currently not known whether their profile/amount varies with ontogenesis in the airways.

The purpose of this study is to further uncover mechanisms governing the lack of spontaneous relaxation in immature-hyperresponsive ASM. We aimed at answering the following questions: 1) Do prostanoids play a predominant role in the ontogenesis of ASM spontaneous relaxation compared with other metabolites of arachidonic acid such as leukotrienes? 2) Which of the COX isoforms is (or both are) responsible for the regulation of spontaneous relaxation? 3) Does the release of prostanoids vary at different maturational stages? We dealt with the first question by measuring the effect of lipoxygenase inhibition and cysteinyl leukotriene receptor-1 antagonism on ASM spontaneous relaxation in guinea pig tracheal strips of different ages. The second question was answered by measuring spontaneous relaxation in the presence of selective inhibitors to COX-1 or to COX-2. The third question was addressed by measuring, using radioimmunoassay, the release of prostanoids in tracheal segments obtained from 1-wk-old, 3-wk-old, and adult guinea pigs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and ASM preparation. Hartley guinea pigs (Charles River Laboratories, Wilmington, MA) were employed for this investigation according to a protocol approved by the Duke University Institutional Animal Care and Use Committee. Animals were housed following the institutional policy of environmental enrichment, which regulates cage environment aiming at enhancing the animal well being. Three age groups were selected based on previous studies from our laboratory: 1-wk-old, as infant guinea pigs [1 wk, 6.7 ± 0.8 (means ± SD)-day-old, 141.5 ± 21.4 g, n = 31], 3-wk-old, as juvenile guinea pigs (3 wk, 21.3 ± 1.8-day-old, 229.8 ± 26.2 g, n = 19), and 2- to 3-mo-old, as adult guinea pigs (adult, 85.0 ± 17.8-day-old, 741.5 ± 94.2 g, n = 31). The tracheas were obtained as previously described (8, 10). Briefly, animals were anesthetized with 200 mg/kg ip pentobarbital sodium. Upon complete achievement of anesthesia, which was confirmed by absence of reflex in response to toe clamping, trachea and lungs were exposed, excised, and immediately immersed in Krebs-Henseleit buffer solution (K-H). The composition of the K-H was the following (mM): 115 NaCl, 25 NaHCO3, 1.38 NaH2PO4, 2.5 KCl, 2.46 MgSO4, 1.9 CaCl2, and 11.2 dextrose, aerated with 95% O2 and 5% CO2. After cleaning away loose connective tissue, tracheal strips were obtained for measurements of spontaneous relaxation, and tracheal segments were obtained for measurements of prostanoid release. All the preparation procedures were performed in K-H solution buffered to pH 7.35–7.45 with 95% O2/5% CO2.

Spontaneous relaxation. As previously described (8, 10), cartilage rings were cut ventrally along the longitudinal axis of the trachea. Strips ~1-mm wide with ~2-mm cartilaginous attachments at both ends were dissected from transverse sections of the trachea such that the ASM was parallel-oriented along the longitudinal axis of the strips. Care was taken to preserve the integrity of the epithelium, since we had previously shown that it contributes to spontaneous relaxation (8), and our aim was to compare relaxation at different ages in a setting analogous to the in vivo physiological condition. One cartilaginous end was clamped in a stainless steel clip at the bottom of an 80-ml double-jacketed organ bath with 37°C K-H prepared as above. The other end was fixed to the transducer tip of an electromagnetic lever system with 4-0 braided silk surgical thread inserted through the cartilage, such that the two cartilage pieces constituted the holders of the muscle via their natural structural connections.

After equilibrating the tracheal strips by leaving them at rest in warm K-H solution for 60 min, supramaximal electric field stimulation (EFS; 18 V, 60 Hz, 400 mA/cm2) was performed using platinum electrodes. This setting of the stimulation was found in preliminary tests to induce maximal contractile response in our preparations. The study was performed with a computerized electromagnetic lever system modified from its original design (5) (Qjin Design, Winnipeg, MB, Canada) as previously described (8). Its force-displacement transducer had a resolution of 0.1 mN and a controllable load ranging between 0 and 150 mN. The voltage signal from the transducer was converted into a digital signal by an RTD1000 computer board (Real Time Device, State College, PA), and data acquisition and analysis were performed by a dedicated computer program (Cunningham Engineering, Lethbridge, AB, Canada).

Strips were first stretched and adapted to a length at which the maximal force was produced in response to EFS. Then the effect of lipoxygenase inhibition, cysteinyl leukotriene receptor-1 antagonism, COX-1 inhibition, or COX-2 inhibition on contractile and relaxation responses was studied. To characterize the contractile function, we measured the following parameters (for detailed definitions of each parameter see Ref. 8): the basal tension in the absence of stimulation, resting tension (RT); the maximal active tension generated in response to EFS (ATmax); and the maximal total active component of the response, total contraction tension (TCTmax), defined as the sum of ATmax and intrinsic tone (the active component of RT). TCTmax was calculated as the difference between maximal total tension measured during a contraction and passive resting tension measured 30–40 s after the removal of stimulation. These parameters were expressed as stress by normalizing force values to the cross-sectional area of the strips. The cross-sectional area was calculated as the product of width and thickness, measured with a VK-C370 digital signal processor Hitachi video camera (Hitachi Home Electronics, Norcross, GA). To evaluate spontaneous relaxation, EFS of 20-s duration was applied and the response recorded. This duration of the EFS was found to induce a substantial spontaneous relaxation in guinea pig tracheal strips (8). To quantify spontaneous relaxation, we used three parameters. The tension relaxation at the end of EFS (TRend) was developed as a more direct expression of the extent of relaxation attained at the end of 20-s EFS than the equivalent parameter TCTend used in our previous study (8). The maximal rate of tension relaxation during stimulation (RTRst) was used as an index of the rate with which active tension drops off during the EFS. Both parameters were normalized to the maximal potential tension drop TCTmax. As an index of the relaxation after removal of the stimulus, we used t1/2,TCTend, the time required to reduce the residual contractile tension at the end of the EFS (TCTend) by one-half. Both RTRst and t1/2,TCTend were previously defined (8).

To assess the role of endogenous leukotrienes in spontaneous relaxation, tracheal strips were incubated with nordihydroguaiaretic acid (NDGA; Sigma-Aldrich, St. Louis, MO), an inhibitor of lipoxygenase, or MK571 (Cayman Chemicals, Ann Arbor, MI), an antagonist of cysteinyl leukotriene receptor-1. NDGA or MK571 was dissolved in ethanol and added to the tissue bath at increasing concentrations (3 x 10–8 to 3 x 10–5 and 10–9 to 10–6 M, respectively). To assess the role of COX-1 and COX-2 individually, tracheal strips were incubated in SC560 (specific inhibitor of COX-1, Cayman Chemicals) or CAY10404 (specific inhibitor of COX-2, Cayman Chemicals), respectively. SC560 or CAY10404 was dissolved in ethanol and added to the tissue bath at increasing concentrations (10–8 to 10–5 M). At each concentration of the inhibitors, ~25-min incubation time was allowed before a 20-s EFS was elicited to measure spontaneous relaxation. During the incubation time, three 10-s EFS were elicited at an interval of 6 min to maintain and monitor the contractility of the strips. The amount of ethanol needed to complete the cumulative concentration-response to NDGA (1.2 µl/ml), MK571 (1.1 µl/ml), SC560 (0.5 µl/ml), and CAY10404 (0.5 µl/ml) had no effect on the muscle strips per se and did not affect either the contractile or the relaxing response to EFS.

Prostanoid release. Tracheal segments obtained by splitting the trachea transversally into two halves were weighed and put into K-H solution aerated with 95% O2 and 5% CO2 at 37°C. To measure prostanoid release, one-half trachea was incubated in 2 ml of K-H solution for 40 min and then stimulated electrically (18 V, 60 Hz, 20 s) six times at 6-min intervals. The second half was maintained in 2 ml of K-H solution without any EFS for 70 min. This incubation protocol was chosen to mimic the experimental conditions under which ASM spontaneous relaxation was measured in tracheal strips. The incubating fluid was then separately collected, divided into aliquots, and frozen in liquid N2 for subsequent prostanoid measurement. In a separate set of experiments, the epithelium was removed by gently scraping the inner side of tracheal segments with the smooth edge of curved forceps. To prepare adequate standard curves for each of the metabolites to be studied, K-H without tracheal segments was treated following the same protocol used for tracheal samples: either 70 min aerated with 95% O2 and 5% CO2 at 37°C or 40 min in the same conditions followed by EFS six times at 6-min intervals. Standards with known concentrations of each specific prostanoid (Biomol Research Labs, Plymouth Meeting, PA) were obtained in prepared K-H. The concentrations of prostanoids released by tracheal segments were measured by radioimmunoassay against the standard curve. We measured TXB2 (stable metabolite of TXA2), 6-keto-PGF1{alpha} (stable metabolite of PGI2), PGE2, and PGF2{alpha}. Samples (measured in duplicate) and standards (measured in triplicate) were incubated with known quantities of [3H]-prostanoid (New England Nuclear, Boston, MA) and polyclonal antisera (Amersham Biosciences, Piscataway, NJ) at 4°C for 24 h. After the incubation, eicosanoids not bonded to the antisera were removed from the mixture with a suspension of dextran-coated activated charcoal. Eicosanoids bonded to the antisera were then detected by counting the radioactivity in the samples/standards with a scintillation counter. The concentration of a prostanoid in each sample was then calculated by interpolation of the obtained standard curve and expressed as picograms/milligrams of fresh tracheal tissue.

Statistics. Data are expressed as means ± SE, except when differently indicated. Statistical analysis was carried out using SigmaStat (version 3.5; Systat, San Jose, CA). The effect of NDGA, MK571, SC560, and CAY10404 on contraction and relaxation at various concentrations was analyzed using repeated measures general ANOVA. Multiple comparisons vs. control group without inhibitors or antagonist were performed using Holm-Sidak method. Age differences in prostanoid release under basal conditions and following EFS were analyzed using general ANOVA. Values of P < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Maturation of ASM spontaneous relaxation. The experiments performed for the present study confirmed our previously published data (8). As in our previous study, there was no statistically significant age difference in TCTmax, the contractile parameter used to normalize relaxation indices. Its value was, respectively, 27.3 ± 2.3, 27.4 ± 1.7, and 29.6 ± 2.1 mN/mm2 in 1-wk, 3-wk, and adult.

Figure 1 shows representative tracings of response to 20-s EFS in each of the three age groups. Similar to our previous observations (8), spontaneous relaxation during EFS increased significantly with age. We found a substantial relaxation during the course of 20-s EFS in adult strips, a moderate relaxation during stimulation in 3 wk, and an almost absent relaxation in 1 wk. As shown in Fig. 2, 1-wk strips relaxed less than 10% TCTmax, whereas adult strips relaxed by ~25% TCTmax. There was a statistically significant increase in TRend (P < 0.001) in 3-wk and adult groups compared with 1-wk. Similar to our previous findings (8), the RTRst was significantly increased in 3-wk (–0.031 ± 0.005 TCTmax/s) and adult (–0.035 ± 0.005) compared with 1-wk (–0.017 ± 0.003). The extent of spontaneous relaxation in adult strips was equivalent to ~50% of the maximal active tension generated in response to the EFS.


Figure 1
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  		Fig. 1. Typical tracings of the response to 20-s electric field stimulation (EFS) in tracheal strips from 1-wk-old (1 wk), 3-wk-old (3 wk), and adult guinea pigs. The 2 parameters used to study the relaxation during EFS are illustrated in the adult panel: TRend, tension relaxation at the end of stimulation; RTRst, maximal rate of tension relaxation. Adult animals showed substantial spontaneous relaxation, which was almost absent in 1-wk animals.

 

Figure 2
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  		Fig. 2. Spontaneous relaxation in tracheal strips from 1-wk (n = 21), 3-wk (n = 10), and adult (n = 21) guinea pigs during a 20-s EFS. Data represent the means and SE. Residual tension at the end of stimulation (TRend) was expressed as % maximal total contraction tension (TCTmax). *Relaxation was significantly less in 1-wk animals compared with 3-wk and adult animals (P < 0.05).

 
The index of relaxation after the stimulation was turned off, t1/2,TCTend, showed constant values across the three age groups. The means ± SE of the three age groups were: 1-wk, 3.79 ± 0.09 s; 3-wk, 3.73 ± 0.11; and adult, 3.93 ± 0.10.

Role of leukotrienes in spontaneous relaxation. To evaluate the role of leukotrienes, we compared EFS-induced spontaneous relaxation in the same tissue preparations before and after treatment with either the lipoxygenase inhibitor NDGA or the cysteinyl leukotriene receptor-1 antagonist MK571.

The effects of the highest concentration of NDGA or MK571 on ASM tone at rest and following EFS are shown in Table 1. NDGA had no effect until its concentration reached 3 x 10–5 M, the maximal concentration used in this study. By contrast, MK571 had a concentration-dependent effect on ASM tone. Nonetheless, NDGA and MK571 caused the same qualitative effects: increase of ATmax and reduction of RT and TCTmax.


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  		Table 1. Effect of NDGA and MK571 on airway smooth muscle tone at rest and in response to 10-s electric field stimulation in guinea pig tracheal strips

 
During 20-s EFS, NDGA did not affect relaxation until the highest concentration used in this study. At 3 x 10–5 M, it significantly reduced both markers of spontaneous relaxation, TRend and RTRst (P < 0.01, Fig. 3). However, it did not reduce the maturational trend observed under basal conditions, i.e., the spontaneous relaxation increased with age. MK571 had no effect on spontaneous relaxation during EFS (Fig. 4).


Figure 3
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  		Fig. 3. Effect of nordihydroguaiaretic acid (NDGA), 3 x 10–5 M, on total tension relaxation TRend and maximal rate of tension relaxation RTRst measured from response to 20-s EFS. A: TRend measured at the end of the stimulation and expressed as % maximal total contraction tension (TCTmax). B: RTRst expressed as TCTmax/s. Data represent the means and SE; n = 5 for each age group. 3 x 10–5 M NDGA significantly reduced both TRend and RTRst (P < 0.01), but it did not reduce the age differences in spontaneous relaxation.

 

Figure 4
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  		Fig. 4. Effect of 10–6 M MK571 on total tension relaxation TRend and maximal rate of tension relaxation RTRst measured from response to 20-s EFS. A: TRend measured at the end of the stimulation and expressed as % maximal total contraction tension (TCTmax). B: RTRst expressed as TCTmax/s. Data represent the means and SE; n = 5 for each age group. MK571 did not affect spontaneous relaxation in either age group.

 
Relaxation after the end of 20-s EFS was not affected by NDGA or MK571, as reflected in the unaltered values of t1/2,TCTend in any age group. The means ± SE in the absence and in the presence of 3 x 10–5 M NDGA were, respectively, 3.7 ± 0.2 and 3.9 ± 0.2 s in 1-wk, 3.5 ± 0.1 and 3.6 ± 0.1 in 3-wk, and 4.0 ± 0.3 and 4.1 ± 0.2 in adult, whereas in the absence and in the presence of 10–6 M MK571 they were, respectively, 3.4 ± 0.2 and 3.4 ± 0.2 in 1-wk and 3.8 ± 0.2 and 3.6 ± 0.2 in adult.

These data show that lipoxygenase inhibition reduced ASM tone at rest but facilitated maintenance of elevated ASM tone in the course of stimulation, thus reducing the extent of spontaneous relaxation. Antagonism of the cysteinyl leukotriene receptor-1 had a similar effect on ASM tone but did not affect spontaneous relaxation. On the one hand, this shows that the reduced spontaneous relaxation by NDGA is not mediated by cysteinyl leukotrienes. On the other hand, this suggests that while leukotrienes may participate in the generation of ASM tone at rest, they do not contribute to the reduced ability of ASM to spontaneously relax during contractile stimulation in immature animals.

Role of COX-1 and COX-2 in spontaneous relaxation. To evaluate the individual role of COX-1 and COX-2, we compared EFS-induced spontaneous relaxation in the same tracheal strips before and after treatment with the specific inhibitors SC560 and CAY10404, respectively. We examined only 1-wk and adult groups because these two groups displayed the most pronounced age difference under basal conditions.

The effects of the highest concentration of SC560 or CAY10404 on ASM tone at rest and following EFS are shown in Table 2. No age-related difference was introduced by either SC560 or CAY10404 in response to 10-s EFS. SC560 had no effect on ATmax at any concentration in either of the two age groups. At the concentration of 10–5 M, CAY10404 substantially reduced ATmax and TCTmax in both 1-wk and adult.


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  		Table 2. Effect of SC560 and CAY10404 on airway smooth muscle tone at rest and in response to 10-s electric field stimulation in guinea pig tracheal strips

 
In response to 20-s EFS, SC560 had no effect on TRend or RTRst in either age group or at any concentration when compared with the same strips under basal conditions. CAY10404, on the other hand, increased both TRend and RTRst significantly at 10–5 M. Figure 5 shows typical tracings of response to 20-s EFS from a 1-wk strip under basal condition and in the presence of CAY10404 (10–5 M). At baseline, no spontaneous relaxation was observed. In the presence of the COX-2 inhibitor CAY10404, 1-wk strips relaxed with substantial TRend and RTRst similar to the typical tracings obtained from adult strips under basal conditions (Figs. 1 and 2). When compared with basal conditions in the same strips, the increase in TRend and RTRst was significant in both age groups (P < 0.001). Specifically, TRend and RTRst in 1-wk reached a level slightly higher than untreated adult ASM. In adult, spontaneous relaxation almost completely reversed the contraction (Fig. 6). However, treatment with CAY10404 at any concentration did not abolish the age difference between 1-wk and adult groups. When CAY10404 was added to the tissue bath in addition to SC560 (10–5 M), the same enhancement of TRend and RTRst was observed, showing that the two COX isoforms do not interact to affect ASM relaxation and confirming that it was the CAY10404 and not SC560 that increased spontaneous relaxation.


Figure 5
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  		Fig. 5. Typical tracings of a 1-wk tracheal strip in response to a 20-s EFS. Top: under basal conditions without inhibitor. Bottom: in the presence of COX-2 inhibitor CAY10404 (10–5 M). TRend, tension relaxation at the end of stimulation, and RTRst, maximal rate of tension relaxation, are illustrated at bottom. Spontaneous relaxation was induced in 1-wk strips after COX-2 inhibition.

 

Figure 6
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  		Fig. 6. Effect of CAY10404 (10–5 M) on total tension relaxation TRend and maximal rate of tension relaxation RTRst measured from response to 20-s EFS. A: TRend measured at the end of the stimulation and expressed as % maximal total contraction tension (TCTmax). B: RTRst expressed as TCTmax/s. Data represent the means and SE; n = 6 for each age group. *In the presence of CAY10404, relaxation was increased in both age groups but was still significantly less in 1-wk than in adult (P < 0.001).

 
Relaxation after the end of 20-s EFS, t1/2,TCTend, was not affected by SC560 at any concentration used in this study. However, at 10–5 M, CAY10404 significantly shortened t1/2,TCTend in both 1-wk (P < 0.001) and adult (P < 0.001) groups (Fig. 7).


Figure 7
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  		Fig. 7. Effect of CAY10404 (10–5 M) on relaxation after the end of EFS, t1/2,TCTend, measured from response to 20-s EFS. Data represent the means and SE; n = 6 for each age group. In the presence of CAY10404, t1/2,TCTend was significantly shortened in both 1-wk (P < 0.001) and adult (P < 0.001).

 
These results show that inhibition of COX-1 with SC560 did not affect contractile or relaxation parameters, but inhibition of COX-2 with CAY10404 (10–5 M) reduced ATmax during 10-s EFS; increased TRend and RTRst during 20-s EFS; and reduced t1/2,TCTend after 20-s EFS. It is COX-2 but not COX-1 that is responsible for the reduction in the extent and the rate of the spontaneous relaxation during the course of EFS.

Ontogenesis of prostanoid release from tracheal segments. Because prostanoids may reduce spontaneous relaxation in immature ASM (8), the release of 6-keto-PGF1{alpha}, PGE2, PGF2{alpha}, and TXB2 was measured in the incubation fluid of tracheal segments. Both the basal (spontaneous) release from tracheal segments without contractile stimulation and the release following EFS showed prominent maturational trends. Each of these prostanoids was the most abundant in the incubation fluid from 1-wk tracheal segments and declined as the animals matured to adulthood (Figs. 8 and 9, P < 0.01). Figure 8 shows the spontaneous and EFS-induced release of the contractile prostanoids TXB2 and PGF2{alpha}. Figure 9 shows the spontaneous and EFS-induced release of the relaxing prostanoids 6-keto-PGF1{alpha} and PGE2. TXB2 was measured as an index of TXA2 release, whereas 6-keto-PGF1{alpha} was measured as an index of PGI2 release. TXB2 was found to be the most abundant in all age groups. EFS induced a significant increase in the release of 6-keto-PGF1{alpha} (P < 0.05) and PGF2{alpha} (P < 0.05) and a slight but not statistically significant increase of the release of TXB2. However, the increase in prostanoid release due to EFS was not statistically different among age groups, hence EFS did not alter the maturational profile of each type of prostanoids. These data suggest that differences in basal release of prostanoids may be involved in the modulation of ASM spontaneous relaxation.


Figure 8
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  		Fig. 8. Thromboxane B2 (TXB2) and prostaglandin F2{alpha} (PGF2{alpha}) measured by radioimmunoassay from incubation fluids of tracheal segments from 1-wk (n = 5), 3-wk (n = 4), and adult (n = 5) guinea pigs. Of each trachea, one-half was kept unstimulated in warm Krebs-Henseleit (K-H) solution (spontaneous release, open bar), whereas the other half underwent periodic EFS (EFS-induced release, hatched bar). Data represent the means and SE in pg/mg fresh tracheal tissue. The release of TXB2 and PGF2{alpha} was the most abundant in the incubation fluid from 1-wk tracheal segments and declined as the animals matured to adulthood (P < 0.05 and P < 0.01, respectively).

 

Figure 9
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  		Fig. 9. 6-Keto-prostaglandin F1{alpha} (6-keto-PGF1{alpha}) and prostaglandin E2 (PGE2) measured by radioimmunoassay from incubation fluids of tracheal segments from 1-wk (n = 5), 3-wk (n = 4), and adult (n = 5) guinea pigs. Of each trachea, one-half was kept unstimulated in warm K-H solution (spontaneous release, open bar), whereas the other half underwent periodic EFS (EFS-induced release, hatched bar). Data represent the means and SE in pg/mg fresh tracheal tissue. The release of 6-keto-PGF1{alpha} and PGE2 was the most abundant in the incubation fluid from 1-wk tracheal segments and declined as the animals matured to adulthood (P < 0.001).

 
Similar to that found in tracheas with intact epithelium, the basal release of prostanoids from deepithelialized guinea pig tracheas declined with maturation (Table 3, P < 0.05). We found no reduction in prostanoid release due to removal of epithelium. In fact, the release of 6-keto-PGF1{alpha} and TXB2 was elevated after the removal of epithelium in all groups (P < 0.05). These data suggest that the epithelium is not a major contributor to the released prostanoids in our preparations.


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  		Table 3. Basal release of prostanoids from deepithelialized guinea pig tracheas

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the first part of this study, we evaluated the role of leukotrienes in the maturation of guinea pig ASM spontaneous relaxation. We found that inhibition of leukotriene production actually reduced spontaneous relaxation and did not alter the maturation-specific differences. We also found that antagonism of cysteinyl leukotriene receptor-1 had no effect on spontaneous relaxation. When combined with our previous study in which we showed that inhibition of prostanoid synthesis abolishes age differences by increasing spontaneous relaxation in immature ASM (8), these results suggest that a crucial factor in determining the lack of spontaneous relaxation in the young is the release of prostanoids rather than leukotrienes. The primary enzyme that catalyzes the production of prostanoids is COX, which has COX-1 and COX-2 isoforms. We found that while inhibition of COX-1 had no effect on relaxation, inhibition of COX-2 significantly increased spontaneous relaxation, an effect similar to inhibition of COX using indomethacin as we previously shown (8). This confirms that it is the release of prostanoids, through COX-2 activity, that underlies the hindered spontaneous relaxation during stimulation. We measured the release of prostanoids from guinea pig tracheas and found that their release indeed declines with maturation. This decline can be associated with the progressively increased ability of spontaneous relaxation as the guinea pigs grow into adulthood. We observed that neither administration of EFS nor removal of epithelium altered the maturational profile of prostanoid release. The outcome of this study provides evidence that prostanoid content decreases with age in guinea pig tracheas and supports our postulation that these membrane lipid compounds are key players in the regulation of ASM spontaneous relaxation. Prostanoids may contribute to the role of ASM in the manifestation of airway hyperresponsiveness during early stages of ontogenesis.

As outlined in the introduction, prostanoids and leukotrienes are respectively generated by COX and lipoxygenase from the common substrate arachidonic acid (20, 47). Both prostanoids and leukotrienes are membrane lipids with proinflammatory properties (36, 40, 46), which exert direct and indirect effects on ASM (2, 6, 23, 36, 52). In our previous study, we investigated the role of prostanoids in ASM relaxation by inhibiting COX with indomethacin (8). In the current study, we evaluated the role of leukotrienes by inhibition of lipoxygenase with NDGA. Both inhibitors reduced the intrinsic tone, a characteristic of guinea pig as well as human ASM. However, they had opposite effects on the ASM ability to spontaneously relax. These data, while confirming that both families of arachidonic acid metabolites contribute to the production of ASM tone at rest (25, 52), suggest that only prostanoids contribute to the reduced relaxation in immature ASM. Our results also imply that the level of intrinsic tone is not a factor in determining spontaneous relaxation and that the action of arachidonic acid metabolites on intrinsic tone and spontaneous relaxation may be exerted through different pathways. It is possible that inhibition of lipoxygenase results in additional production of prostanoids due to increased substrate availability to COX. Indeed, it has been suggested that inhibition of COX could "shunt" arachidonate to the lipoxygenase pathway (17, 28) and vice versa (1, 31). Since leukotrienes are known to exert mainly contractile effects on ASM, the reduced relaxation we observed cannot originate directly from the reduced amount of leukotrienes due to inhibition of their synthesis. In our experiments, a shunt effect could be one of the possible scenarios, in which the increased prostanoid generation would lead to the reduced relaxation produced by NDGA. The lack of reduced relaxation by antagonizing cysteinyl leukotriene receptor-1, which would not affect the tissue content of prostanoid, further supports this possibility. Alternatively, this reduced relaxation could be determined by an effect of NDGA on a target other than leukotriene synthesis. Regardless, the important message we gain from this part of our study is that inhibition of leukotriene synthesis or antagonism of cysteinyl leukotriene receptor-1 did not enhance relaxation, indicating that the lipoxygenase pathway is not involved in the reduced ability of infant airways to spontaneously relax compared with mature airways.

COX, on the other hand, is implicated in spontaneous relaxation (8). The results of the current study further suggest that, of the two isoforms of COX, it is the inducible form COX-2 that regulates spontaneous relaxation. Conventionally, COX-1 has been thought to be expressed constitutively in most tissues and serves predominantly homeostatic functions. COX-2 was thought to be mostly expressed in monocytes, neutrophils, macrophages, and endothelial cells and primarily involved with pathological processes such as inflammation and tumorigenesis. Indeed, COX-2 but not COX-1 has been found to be significantly upregulated during inflammatory responses such as after allergic sensitization (38) and chronic exposure to air pollution, ozone, and diesel particles (7). However, recent views on COX-2 have been evolving such that its role in normal physiology and wound healing has been increasingly recognized. COX-2 serves distinct functions such as ovulation, implantation, and resolution of inflammation (18, 34). Recently, COX-2 has been found to be expressed in various healthy tissue and organs, even in the same cellular compartments as COX-1 (44). A study in human airways detected COX-2 mRNA by in situ hybridization and protein by immunohistochemistry in the epithelium in the absence of airway inflammation (50). The same study also found measurable levels of COX-2 mRNA and proteins in cultured epithelial cells not exposed to inflammatory stimulation. It is therefore possible that COX-2 expression may be substantial even in the absence of specific induction. It is also possible that COX-2 present in the airway epithelium, which was not removed from our preparations, contributes to the regulation of the extent and rate of ASM spontaneous relaxation. Relevant to our study, COX-2 but not COX-1 has been shown to be essential in neonatal development, e.g., by determining the normal glomerular formation (24) and the closure of the ductus arteriosus (26). Furthermore, a neonatal maturation of COX-1 and COX-2 gene expression and protein content has been shown in ovine peripheral lung (4). Similar ontogenetic changes are likely to take place in the airways and in the ASM and will need further studies. Our results provide the first evidence that COX-2 but not COX-1 is involved in the regulation of the intrinsic ability of ASM to spontaneously reverse its contractile state during stimulation. This suggests that the lack of relaxation in newborn animals could be due to pronounced activity of COX-2 during this stage of ontogenesis. Although its physiological advantage remains to be investigated, this particular ontogenetic feature of COX-2 is associated with a pronounced contractile state of ASM due to diminished spontaneous relaxation, which is consistent with the prevalent airway hyperresponsiveness that occurs in the immature stage of life.

It is interesting that the specific inhibition of COX-2 with CAY10404 did not reduce the age difference as did the inhibition of COX with indomethacin (8), but determined a greater increase of relaxation than indomethacin in both age groups. The different effect of the two inhibitors on adult tissue could be explained by the difference in specific affinity of the inhibitors to the targeted isoforms. The IC50 of indomethacin to COX-2 is 6 µM, whereas CAY10404 has an IC50 to COX-2 less than 1 nM. Also, CAY10404 has a selectivity index (IC50 COX-1/IC50 COX-2) greater than 500,000, whereas the selectivity index of indomethacin is 0.017, making CAY10404 the most selective COX-2 inhibitor currently available. It is possible that the inhibitory action of indomethacin may not be sufficient to completely abrogate the activity of COX-2 in ASM. We also found that the increase in relaxation in the presence of CAY10404 was more profound in adult (reaching almost 80% TCTmax) than in 1-wk (reaching ~40% TCTmax) (Fig. 6). This may suggest that although COX-2 may be the main factor regulating spontaneous relaxation in adult ASM, it may not be the only regulator in 1-wk ASM. Factors other than COX-2 could also contribute to the hindered spontaneous relaxation in ASM of immature guinea pig airways.

The other main finding of the present study was that the release of prostanoids from tracheal tissue followed an ontogenetic trend consistent with the ASM ability to spontaneously relax. We found that the most abundant prostanoid release occurred in the 1-wk or immature age group, in which spontaneous relaxation is almost absent. Although the relative release of different types of prostanoids was similar in all age groups, contractile prostanoids were released in the greatest quantities according to the following order: TXB2 > PGF2{alpha} > 6-keto-PGF1{alpha} > PGE2. After the tissue was electrically stimulated, the release of prostanoids was slightly increased at all ages but without any alteration of their relative proportions. These data suggest that it is the greater amount of prostanoids released at rest that modulates spontaneous relaxation. Considering that plasma prostaglandin levels have been shown to be elevated in human during early neonatal life (32), our results seem to have potential relevance to the ontogenesis of human physiology. Since both contractile and relaxing prostanoids are more abundant in immature ASM, it is possible that other factors are also involved in the maturation of spontaneous relaxation, e.g., density and/or distribution of the different prostanoid receptors. Indeed, prostanoid receptors have been shown to undergo ontogenetic changes in smooth muscle from the brain and ocular vasculature. However, these changes seem to occur in an organ- and receptor-specific fashion, with EP and FP receptors mostly increasing toward adulthood, whereas IP and TP receptors decrease in certain microvessels and increase in others (51). Although this variability in the maturational changes of prostanoid receptors prevents a prediction of the ontogenetic pattern of prostanoid receptors in ASM, it does suggest that maturational changes in receptor density/distribution are also likely to occur in ASM and in other airway cell types. Because the ontogenesis of prostanoid receptors and of the response to exogenous prostanoids in guinea pig ASM is currently unknown, the resolution of this issue would need further investigation.

Epithelium has been considered as one of the major sources of prostanoid production in the airways (21, 29). However, our study shows that removal of epithelium from guinea pig tracheas did not induce an expected reduction of prostanoid release. On the contrary, the release of 6-keto-PGF1{alpha} and TXB2 was even elevated. Although the reason for this elevation remains unclear, the increase of these contractile prostanoids can contribute to the reduced spontaneous relaxation in deepithelialized ASM preparations (8).

Finally, here we discuss the relevance of our findings to airway hyperresponsiveness. Increased shortening of ASM may contribute to the onset and degree of bronchoconstriction, but may not fully explain the sustained bronchoconstriction. Currently, the universally adopted therapy to reverse bronchospasm is the induction of ASM relaxation with β-adrenergic agonists. However, a reduced ability to relax in response to these agonists has been shown in postmortem samples of tracheal smooth muscle from asthmatic subjects (3, 19). Similarly, in vitro passive sensitization of human bronchi, achieved by incubation in serum containing high levels of IgE, reduced ASM relaxant response to β2-agonists compared with control tissue (43). These findings suggest that exogenous relaxant agents are less effective in asthmatics than in healthy subjects. Nonetheless, studies on pharmacologically induced relaxation fail to elucidate whether the ability of ASM to spontaneously relax is intrinsically altered in airway hyperresponsiveness. The existing studies on spontaneous relaxation in ASM from hyperresponsive animals, although very few, have clearly shown that this property is impaired in hyperresponsive airways. Tracheal smooth muscle from allergic dogs, characterized by airway and ASM hyperresponsiveness, does not exhibit the typical relaxation shown by littermate controls during EFS (33). Likewise, at early stages of ontogenesis, when greater airway responsiveness occurs compared with adults (22, 35, 41, 45), the inhibitory control of ASM tone is not well developed (8, 13). Most recently we showed that spontaneous relaxation is markedly absent in A/J mice, a strain characterized by airway hyperresponsiveness, whereas a substantial relaxation is present in the C57BL/6J mice, a strain that is characterized by normal airway responsiveness (11). The accumulating evidence from separate animal models suggests that airway hyperresponsiveness could be at least in part determined by a reduced ability of ASM to spontaneously relax and the subsequent impairment in spontaneous bronchodilatation. Our findings suggest that prostanoids may be critical mediators responsible for reduced spontaneous relaxation in immature ASM and that these mediators may play a central role in the occurrence of airway hyperresponsiveness in the young. Whether prostanoids also play a role in the removal of bronchospasm in asthmatics remains to be investigated.

In conclusion, we showed that leukotrienes do not play a role in inhibiting spontaneous relaxation in guinea pig ASM. On the other hand, we confirmed that inhibition of prostanoid release by specifically inhibiting COX-2 increases the ability of ASM to spontaneously relax during the course of contractile stimulation. Prostanoids are most abundantly released during early postnatal life in guinea pig tracheal tissue, and this release decreases significantly and progressively with ontogenesis. Since we showed previously as well as in this study that inhibition of prostanoid synthesis increases spontaneous relaxation in ASM from infant guinea pigs to levels normally occurring in adult ASM, we suggest that the naturally high prostanoid levels during the early stages of development is responsible for the reduced ASM spontaneous relaxation and consequently plays a role in airway hyperresponsiveness in the young.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-075307, HL-48376, and HL-61899, an American Lung Association Biomedical Research Grant, an American Lung Association of North Carolina CS Venable Award, and by Duke Children's Miracle Network research grants.


   ACKNOWLEDGMENTS
 
This work was partially presented in preliminary form at the 2001 World Asthma Meeting and at the 2002 and 2008 Meetings of the American Thoracic Society.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Chitano, Dept. of Pediatrics, Duke Univ. Medical Center, Rm. 302, Bell Bldg., Box 2994, Durham, NC 27710 (e-mail: chita001{at}mc.duke.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
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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