Airway synthesis of 20-hydroxyeicosatetraenoic acid: metabolism by cyclooxygenase to a bronchodilator

Elizabeth R. Jacobs1,2, Richard M. Effros2, John R. Falck3, K. Malla Reddy3, William B. Campbell4, and Daling Zhu1

1 Department of Physiology, Cardiovascular Research Center; 2 Department of Medicine; and 4 Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; and 3 Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235


    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Rabbit airway tissue is a particularly rich source of cytochrome P-4504A protein, but very little information regarding the effect(s) of 20-hydroxyeicosatetraenoic acid (20-HETE) on bronchial tone is available. Our studies examined the response of rabbit bronchial rings to 20-HETE and the metabolism of arachidonic acid and 20-HETE from airway microsomes. 20-HETE (10-8 to 10-6 M) produced a concentration-dependent relaxation of bronchial rings precontracted with KCl or histamine but not with carbachol. Relaxation to 20-HETE was blocked by indomethacin or epithelium removal, consistent with the conversion of 20-HETE to a bronchial relaxant by epithelial cyclooxygenase. A cyclooxygenase product of 20-HETE also elicited relaxation of bronchial rings. [14C]arachidonic acid was converted by airway microsomes to products that comigrated with authentic 20-HETE (confirmed by gas chromatography-mass spectrometry as 19- and 20-HETE) and to unidentified polar metabolites. [3H]20-HETE was metabolized to indomethacin-inhibitable products. These data suggest that 20-HETE is an endogenous product of rabbit airway tissue and may modulate airway resistance in a cyclooxygenase-dependent manner.

bronchi; bronchodilation; eicosanoid; arachidonic acid; cytochrome P-450


    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

FOR MORE THAN 20 years, the capacity of pregnant rabbit lungs to metabolize arachidonic acid (AA) into 20-hydroxyeicosatetraenoic acid (20-HETE) has been recognized (27). Birks et al. (1) reported the expression of cytochrome P-450 (CYP)4A proteins and 20-HETE synthesis in microsomal proteins of human peripheral lung tissue and demonstrated concentration- and cyclooxygenase (COX)-dependent dilation of small pulmonary arteries by 20-HETE. These data extended the expression of CYP4A to human lungs and raised the possibility that CYP4A products may modulate pulmonary vascular tone. Immunohistochemical studies of CYP4A4 in rabbit lungs suggested that CYP4A protein is present in nonciliated cells of the proximal airways as well as in end capillary endothelial cells (21). Our studies demonstrated abundant immunospecific protein localization and formation of AA metabolites that coelute with authentic 20-HETE in rabbit airway tissue above those of peripheral lung and vascular microsomes (41). These observations raise the suggestion that CYP4A products could have a role in the modulation of airway resistance. However, no systematic investigation of the CYP products in the airway or studies of the physiological role for these products in control of airway tone were available. Our investigations were designed to first determine the effect of 20-HETE and its COX metabolite(s) on the tone of rabbit bronchial rings. Second, we endeavored to establish the identification of omega -hydroxylase metabolites of AA metabolites of airway tissue that comigrate with authentic 20-HETE by gas chromatography (GC)-mass spectrometry (MS). In related experiments, we sought evidence for COX metabolites of AA and [3H]20-HETE in airway microsomes. These studies were undertaken because we found indomethacin-inhibitable effects of 20-HETE on airway tone and because of evidence that 20-HETE may serve as substrate for COX in other systems (16). Third, we examined CYP4A expression in airway tissue using a semiquantitative determination of immunospecific protein in sized rabbit tracheae and bronchi. Our data demonstrate a concentration-dependent relaxation of bronchial rings contracted with KCl and histamine to 20-HETE. 20-HETE-induced bronchial relaxation was blocked by removal of airway epithelium or by treatment with indomethacin. A COX metabolite of 20-HETE from airway microsomes caused a concentration-dependent relaxation of bronchial rings. In addition, we confirm the synthesis of 19- and 20-HETE in airway tissue by GC-MS. Both AA and [3H]20-HETE were metabolized by airway microsomes to polar products that were inhibited by the inclusion of indomethacin in the incubation medium. CYP4A and COX-1 immunospecific protein(s) were identified in rabbit airway microsomes. These data are the first to suggest that a COX product of 20-HETE may function as an endogenous modulator of airway tone.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals. Adult male New Zealand White rabbits (weight 2-3 kg) were used in this study, which was approved by our Animal Experimentation Committee and conformed with the recommendations of the Guide for the Care and Use of Laboratory Animals (Washington, DC: Natl. Acad. Press, 1996). The rabbits were cared for in the Animal Resource Center of the Medical College of Wisconsin (Milwaukee), which is approved by the American Association for Accreditation of Laboratory Animal Care. The animals were maintained in our facility for a minimum of 2 days to identify signs of respiratory disease before use in a study.

Preparation of bronchial rings. Bronchial rings were obtained and examined for isometric contractile responses according to the method of Hooker et al. (17). Briefly, the rabbits were anesthetized with intramuscular injections of xylazine (6 mg/kg), ketamine (55 mg/kg), and acepromazine (1.6 mg/kg). The chest was opened, and the lungs were quickly removed and immersed in ice-cold modified Krebs solution, the composition of which was (in mM) 118 NaCl, 4.7 KCl, 27 NaHCO3, 10 glucose, 2.5 CaCl2, 0.57 MgSO4, and 1.2 KH2PO4, and gassed with 95% O2-5% CO2. Connective tissue and adipose tissue were carefully removed, and the right and left main stem bronchi were obtained by teasing away the parenchyma with dissecting forceps, followed by detachment of adhering bronchioles and cutting into 3-mm rings. The epithelium was removed from alternate rings with fine forceps passed through the lumen and gently rotated (epithelium-denuded studies). Histological confirmation of epithelium removal was determined with Movat's pentachrome stain (13) in selected bronchi. The rings were mounted on tungsten wire, one end of which was connected to a fixed holder and the other to a force displacement transducer (model FT03, Gould Electronics), for continuous measurement of isometric tension and were immersed in pH-adjusted, oxygenated Krebs solution at 37°C. Tension data were relayed from the pressure transducers to a signal amplifier (600 series eight-channel amplifier, Gould Electronics). Data were acquired and analyzed with CODAS software (DATAQ Instruments). The rings were initially loaded with 0.75 g of tension that was gradually and incrementally applied over 30 min and then equilibrated for an additional 30-40 min at 37°C in Krebs solution before the studies were begun. The equilibration tension of 0.75 g was chosen based on preliminary studies (data not shown) that demonstrated a maximum contractile response to KCl and carbachol in rings so equilibrated compared with that of rings loaded with 0.5 or 1.0 g of tension.

Protocol for tension studies of bronchial rings. The rings were suspended in a bath containing 25 ml of Krebs solution bubbled with 95% O2-5% CO2 and at 37°C. Contraction of the rings was produced by using 12.5 and 25 mM KCl, 50 µM histamine, or progressive concentrations of carbachol (10-8 to 10-5 M). Relaxation of the contracted rings to incrementally increased concentrations of nifedipine, isoproterenol, and atropine was also examined. Viability of the rings was determined based on brisk contractile and relaxant responses. The baths were then washed, and a submaximal contraction with one of the three agents listed above was established. 20-HETE from a stock solution in ethanol was added to the baths of precontracted rings at a final concentration ranging from 10-8 to 10-6 M at 3-min intervals to determine the concentration-response relationship. Vehicle control experiments with ethanol in twice the greatest concentration utilized under experimental conditions were performed. In some experiments, the COX metabolite of 20-HETE that eluted at 8 min from the C-18 column or blank "control" fraction was collected in 1-min fractions and applied to the rings. After addition of the eicosanoids, the rings were washed by complete exchange of the bath solution three to five times, and KCl was added to the baths again to confirm viability of the rings. In other studies, the rings were preincubated with indomethacin for 30 min before contraction with KCl, and the response to 20-HETE (10-8 to 10-6 M) was tested.

Sizing of microdissected airways for immunospecific protein studies. Preparation of the animals, removal of the lungs, and dissection of the airways were carried out as described in Preparation of bronchial rings. Adventitial tissue surrounding the airways was meticulously removed. For purposes of this study, tracheal tissues (roughly 5-7 mm in outer diameter) and main stem bronchi were considered large airways, third- and fourth-order bronchi were called medium-sized airways (and thus the bronchial rings used for the tension studies were medium in size), and fifth-order and smaller airways (generally <2-mm diameter) were termed small airways. In some experiments, the large airways were opened longitudinally and the epithelium was removed by scraping.

CYP metabolism of AA by airway tissue and microsomes. Microdissected airway tissues were homogenized with a handheld tissue homogenizer (Tissue Tearor model 985-370, Biospec Products). Microsomal proteins were prepared by differential centrifugation with a modification of the methods previously reported by Ma et al. (19) and Birks et al. (1). Protein in the microsomal preparations was quantitated according to the method of Bradford (2). Airway microsomes were resuspended in assay (or incubation) buffer (80 mM K2HPO4, 20 mM KH2PO4, 1 mM EDTA, and 10 mM MgCl2) and incubated at a final protein concentration of 1 mg/ml (200-µl final volume) for 30 min at 37°C with [1-14C]AA (2 µM), 1 mM NADPH, and an NADPH-regenerating system containing 10 mM isocitrate and 0.1 U/ml of isocitrate dehydrogenase (15, 19). Reactions were terminated by acidification with 0.1 M formic acid, and the product was extracted twice with ethyl acetate. The organic phase was back extracted with 1 ml of distilled water, evaporated under nitrogen, and reconstituted in ethanol. Reaction products were separated on a 4.6 × 250-mm C-18 reverse-phase (RP) HPLC column (Supelco, Bellefonte, PA) with a gradient of acetonitrile in water (30-100%) over 50 min and detected with a radioactivity detector [Beckman radioactivity detector model 171; for more details regarding the gradient, see Birks et al. (1)]. Preliminary identification of the products was based on coelution with authentic standards.

In some experiments, as indicated, products first separated by RP HPLC were further analyzed with a normal-phase system (5-µm, 4.6 × 250-mm silica gel column; Beckman Instruments). Solvent A was hexane with 0.1% glacial acetic acid, and solvent B was hexane containing 2% isopropanol and 0.1% glacial acetic acid. The flow rate was 3 ml/min. The HETEs were eluted over 40 min with a linear gradient of 15% solvent B in solvent A to 75% solvent B in solvent A. Absorbance was monitored at 235 and 280 nm with a diode-array detector. Fractions were collected in 0.6-ml fractions for analysis by GC-MS.

GC-MS. Metabolites were separated first by RP HPLC followed by normal-phase HPLC and collected in fractions as previously published by Pfister et al. (25) and Revtyak et al. (29). Eicosanoid products were derivatized to their pentafluorobenzyl (PFB) ester for analysis by negative chemical ionization MS. The eicosanoid in acetonitrile was reacted with alpha -bromo-2,3,4,5,6-pentafluorotoluene in the presence of diisopropylethylamine for 15 min at room temperature. The solvent was dried under nitrogen, and the residue was reconstituted in 2 ml of water and extracted with ethyl acetate. The organic solvent was dried under nitrogen. The hydroxyl groups were converted to their trimethylsilyl (TMS) esters by reaction with 50 µl of bis(TMS)trifluoroacetamide for 1 h at 37°C. The solvent was removed under nitrogen, and the sample was redissolved in acetonitrile. GC-MS analysis was performed with a Hewlett-Packard 5890 series II gas chromatograph and Hewlett-Packard MS Engine quadrupole mass spectrometer. Ionization of the samples was done chemically with CH4 as the reagent gas and detected in the negative-ion mode at 65-60 eV. The GC column was a 30-m DB-5 capillary column. The oven temperature increased from 100 to 300°C over 5 min and was then maintained at 300°C.

Immunospecific protein identification. Microsomal suspensions were separated by electrophoresis on a 8.5 or 10% denaturing sodium dodecyl sulfate-polyacrylamide gel and transferred to a nitrocellulose membrane. Nonspecific binding was blocked by incubating the membrane in Tris-buffered saline in 5 (prostaglandin synthase-1) or 10% (CYP4A) nonfat milk overnight, followed by three washes with Tris-buffered saline. The nitrocellulose membrane was incubated for 2 h at room temperature with a polyclonal antibody to rat liver CYP4A omega -hydroxylase enzyme that cross-reacts with CYP4A1, -4A2, and -4A3 isoforms (15) or a polyclonal antibody to ovine prostaglandin H synthase 1 (COX-1; Cayman Chemical, Ann Arbor, MI). Isoforms CYP4A1, -4A2, and -4A3 have amino acid sequence homology with CYP4A4, -4A6, and -4A7 in the rabbit (30). The membrane was rinsed three times before incubation with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:1,000) and was then visualized with enhanced chemiluminescence. X-ray film was developed on the Kodak XOMAT developer, and the X-ray image of the gel was scanned on a densitometer. Bands corresponding to the 55-kDa molecular-mass marker were selected on the computer representation of the scan, and after background correction, the pixel density within each band was determined by the computer, providing a means for relative quantitation.

Statistics. Data are presented as means ± SE. Tensions of precontracted bronchial rings exposed to three concentrations of 20-HETE were compared by analysis of variance on ranks of repeated measures, followed by Student-Newman-Keuls pairwise comparisons when P values permitted. Differences between immunospecific band density in large- and small-airway microsomes were determined with a two-tailed unpaired t-test. A P value of <= 0.05 by a two-tailed test was considered significant.


    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Bronchial ring studies. Bronchial rings (average weight 8 ± 0.7 mg; n = 12) were initially stretched to a passive tension of 0.75 g. Over the 30-min equilibration period, the rings relaxed to 0.43 ± 0.05 g (n = 14). The maximum increase in tension evoked by KCl was 182 ± 15% of that of baseline tension (n = 14 rings). In rings exposed to KCl, tension increased rapidly (within 1 min) and was stable over at least 15-20 min. In contrast, increases in tension to carbachol were slower to develop, often requiring 5-10 min to reach maximum values, and were of greater magnitude (>9-fold increase in baseline tension in the presence of 10-3 M carbachol; see Fig. 1). Histological examination of representative bronchi confirmed intact epithelium in control experiments and effective removal of epithelium after dissecting forceps were passed through the lumen. Control and deepithelialized rings were selected for study with 20-HETE based on >50% increases in tension on exposure to KCl and/or carbachol. Baseline and percent increases in tension to KCl and carbachol and relaxation to nifedipine and drug washout were not different in control and deepithelialized rings (data not shown). Vehicle alone (ethanol) in the highest concentration used (0.4%) had no effect on ring tension (n = 4).


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Fig. 1.   Responses of rabbit bronchi treated with carbachol. Percentage of baseline tension of 8 rings is plotted as a function of carbachol concentration ([carbachol]) in bath. Carbachol was a potent, reversible bronchoconstrictor, with tension increasing 9- to 10-fold at highest [carbachol].

To assess tension changes in response to 20-HETE, rings were submaximally contracted with KCl, histamine, or carbachol. 20-HETE was then added directly to the bath in increasing concentrations from 10-8 to 10-6 M. A representative response is shown in Fig. 2A. 20-HETE evoked a concentration-dependent relaxation of KCl-contracted rings. In a similar manner, rings preconstricted with histamine (50 µM) exhibited a concentration-dependent relaxation in response to 20-HETE (Fig. 2B). Rings pretreated with indomethacin (1 or 10 µM) or with the epithelium removed exhibited no change in tension when exposed to 20-HETE (see Fig. 2B). Rings pretreated with indomethacin (10 µM), submaximally contracted with KCl, and then exposed to a high concentration of 20-HETE (10-6 M) contracted (147 ± 19% of KCl tension; n = 3; data not shown). In contrast, bronchi exposed to the vehicle for indomethacin followed by 10-6 M 20-HETE exhibited relaxation (79.8 ± 5% of KCl tension; n = 5). The COX metabolite of 20-HETE, with a retention time of ~8 min on our RP HPLC system, elicited a concentration-dependent relaxation of KCl-contracted rings (~5 × 10-11 M, 14% relaxation; 10-10 M, 21% relaxation; n = 2). An approximate molar concentration(s) of this metabolite was estimated by assuming a 1:1 molar conversion of 20-HETE to metabolite and does not reflect recovery correction; thus our calculations represent the upper limit of the possible concentration of the unknown. Negative control fractions eluting from the column 1 min before and 2 min after the 8-min peak of interest, in volumes of ethanol corresponding to the highest concentration of vehicle of the unknown tested (20 µl), had no effect on ring tension (100.4 ± 2% of KCl tension; n = 6).


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Fig. 2.   Responses of rabbit bronchi to KCl and 20-hydroxyeicosatetraenoic acid (20-HETE; 20-H). A: representative record of tensions plotted against time in an airway ring. Ring was first contracted with KCl (initial concentration 12.5 mM, followed by a second dose of 12.5 mM), which elicited a brisk increase in tension from ~0.2 to 0.9 g. After contraction was established, 20-HETE in increasing concentrations was added to bath. A decrease in tension was observed that appeared maximal at 10-7 M 20-HETE. B: averaged responses to 20-HETE of all bronchial rings contracted with KCl or histamine and in rings contracted with KCl after pretreatment with indomethacin (+indomethacin; 10 µM) for 15 min or contracted with KCl after removal of epithelium (-epithelium). [20-HETE], 20-HETE concentration. Data are means ± SE of percentage of contractile (KCl or histamine)-induced tension. 20-HETE effected a concentration-, epithelium-, and cyclooxygenase-dependent relaxation of bronchial rings constricted with KCl or histamine. In data not shown, similar results were observed in rings pretreated with 1 µM indomethacin and exposed to 10 µM 20-HETE (99.7 ± 0.6% contractile tension; n = 4). * Significantly different from -epithelium and +indomethacin.

Rings precontracted with carbachol rather than KCl did not relax with 20-HETE (see Table 1). Baseline and KCl- and/or carbachol-evoked increases in tension of indomethacin-treated or deepithelialized rings were not different from those of control rings (data not shown). Epithelium-denuded rings contracted with acetylcholine (10-7 M) and carbachol (10-7 M) exhibited brisk decreases in tension in response to treatment with nifedipine (10-6 M) and to washout of the drug (n = 2; data not shown). Percent tension increases elicited with KCl (225 ± 24%), carbachol (177 ± 10%), or histamine (337 ± 97%) before the addition of 20-HETE were not different (by ANOVA). Finally, the addition of AA alone (10-8 through 10-6 M) to KCl-contracted rings did not elicit relaxation (Table 1).

                              
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Table 1.   Effect of arachidonic acid on KCl-augmented tension and 20-HETE on carbachol-enhanced bronchial ring tension

CYP assays. A product that migrates with authentic 20-HETE is a dominant metabolite of rabbit airway microsomes incubated with [14C]AA, similar to previous observations by Zhu et al. (41). We routinely observed a relatively broad-based peak on RP HPLC that coelutes around 25 min with [3H]20-HETE (see Fig. 3A). This peak was eliminated by inclusion of the omega -hydroxylase inhibitor 17-octadecynoic acid (data not shown) in the incubation buffer. The retention times of 19- and 20-HETE in our RP liquid-chromatography system are very similar. The mean conversion rate of AA into 19- and 20-HETE was 5.6 ± 1.4 pmol · mg protein-1 · min-1 (n = 11 microsomal preparations). A second polar 14C product that eluted around 8 min was also repeatedly observed. Synthesis of this compound was inhibited by 10 µM indomethacin (see Fig. 3B).


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Fig. 3.   A and B: representative reverse-phase HPLC radiochromatograms of eicosanoid products from microsomal fractions of airway tissue incubated for 30 min with [14C]arachidonic acid (AA; 2 µM) as substrate with vehicle (A) and indomethacin (10-5 M; B) to inhibit formation of cyclooxygenase products. Retention times for authentic standards on this column: 6-keto-PGF1alpha , 6.6 min; PGF2alpha , 11.6 min; PGE, 17 min; 20-HETE, 25.2 min; EETs, 31-32 min; and unmetabolized AA, 38 min. Dominant metabolite of AA is a product that coelutes with 20-HETE (~25 min). A second product (peak 1) is observed at ~8 min. Synthesis of this product is blocked by inclusion of 10 µM indomethacin in assay system. CPM, counts/min. C and D: reverse-phase chromatograms of eicosanoid metabolites of [3H]20-HETE. 20-HETE elutes at ~25 min. Three products with retention times of 8 (peak 1), 16 (peak 2), and 23 (peak 3) min are observed (C). Formation of all 3 of these products is inhibited by indomethacin, consistent with assumption that formation of these metabolites is catalyzed by cyclooxygenase (D). In other experiments (n = 5), product with a retention time of 8 min was consistently observed, whereas those metabolites eluting later were not.

In a separate group of experiments, [3H]20-HETE was incubated with airway microsomes. An example of 3H-labeled eicosanoid products separated by RP HPLC appears in Fig. 3, C and D. Three products are shown, the synthesis of which was inhibited by the inclusion of 10 µM indomethacin in the incubation medium. In other assays performed with [3H]20-HETE as the substrate, the product that eluted at 8 min was consistently observed, whereas the others were variable. The product that elutes at ~8 min has a similar retention time to that of a COX product when AA was used as the substrate (see Fig. 3A). This metabolite (Fig. 3C, peak 1) was tested for effects on bronchial tone because it was reproducibly synthesized and appeared to be a product when both 20-HETE and AA were provided as substrates.

GC-MS studies. To confirm the identity of the 14C-labeled eicosanoid metabolite(s) that comigrated with authentic 20-HETE, 14C products eluting off the C-18 column were collected in fractions. The fraction that contained the 14C metabolite(s) of interest (Fig. 4A, peak 1) was further separated by normal-phase HPLC. Two large peaks that comigrated with authentic 19- and 20-HETE appear in the representative normal-phase chromatogram (Fig. 4B). These products were labeled peaks 2 and 3, respectively. GC-negative-ion chemical ionization MS of the PFB ester of the products that coeluted with authentic 19- and 20-HETE appear in Fig. 4, C and D. Fragments with the expected mass-to-charge ratios of 301 (mass/PFB+TMS) and 391 (mass/PFB), consistent with both 19- and 20-HETE, were observed. Fragments with ratios of 303 and 393 were also noted due to the use of [14C]AA as the substrate.


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Fig. 4.   Gas chromatograph-mass spectrometry identification of 20-HETE in rabbit lung. A: typical reverse-phase chromatogram (similar to those in Fig. 3), with a large peak at ~25 min (peak 1) that coelutes with authentic 20-HETE. Peak 1 was extracted and reanalyzed on a normal-phase HPLC system, which revealed presence of 2 major metabolites. In this normal-phase system, authentic 19-HETE elutes at ~18.4 min and 20-HETE elutes at ~25 min (B, peaks 3 and 2, respectively). Finally, peaks 2 and 3 were collected, derivatized, and subjected to gas chromatography-mass spectrometry. Diagonal lines on x-axis in A indicate expansion (rechromatography) of peak 1 into peaks 2 and 3 in B. C and D: negative-ion chemical mass spectrometry of pentafluorobenzyl esters and/or trimethylsilyl ethers of peaks 3 and 2, respectively, from airway tissue. Fragments with expected mass-to-charge ratio values of 301 and 303 and 391 and 393, consistent with 19- and 20-HETE, respectively, were observed in both mass-spectrum analyses. Cold and uniformly 14C-labeled AA were used as substrates for the reactions, and fragments with masses consistent with 12C and 14C isotopes are therefore evident on mass spectra.

Immunospecific protein studies. Western immunoblots of airway tissue probed with a primary polyclonal antibody raised against rat CYP4A1, -4A2, and -4A3 exhibited an immunospecific band around 55 kDa. Preparations derived from small bronchi (e.g., fifth order and smaller, generally >2 mm) demonstrated less-dense immunospecific bands than those of preparations from larger bronchi from the same animals (P < 0.01; n = 4 rabbits). Duplicate or triplicate samples from each size airway separated and analyzed on the same gel were compared (see Fig. 5A). In some studies, larger airways were opened horizontally, with the epithelium being scraped off from approximately one-half of the sample before homogenization and centrifugation for microsomal protein preparations. In these studies, immunospecific band densities of tissue derived from airways with and without epithelium appear similar (Fig. 5B); relative optical density units of bands from deepithelialized samples were 15% greater than those of paired control samples. Finally, a band of 70 kDa, the expected size for rabbit COX-1 (23), was detected in airway microsomes separated electrophoretically and probed with a primary polyclonal antibody to COX-1.


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Fig. 5.   A: Western blots of microsomal proteins from small, medium, and large airways. Triplicate 10-µg protein aliquots from each sample were separated electrophoretically and probed with a primary antibody raised against cytochrome P-450 (CYP)4A1, -4A2, and -4A3 in rat liver. Immunospecific protein bands at ~55 kDa are clearly seen in all lanes. Bands from large (>4-mm) airways are denser than those from small airways (relative optical density units, 1,613 ± 205 for small and 3,281 ± 458 in large airways; P < 0.04; n = 6). B: representative Western blot probed with CYP4A antibody of protein samples derived from large airways with (+) and without (-) epithelium (epith). Epithelium was removed by scraping. Triplicate samples (10-µg total protein each) from each preparation were studied. Immunospecific bands of approximately similar density are observed in both samples. Relative optical density units from control samples (4,157 ± 52) were less than those from deepithelialized samples (4,788 ± 71; P < 0.01 by paired t-test; n = 8). C: electrophoretically separated proteins from small bronchi (100 µg/lane) of 3 different rabbits probed with a primary antibody against cyclooxygenase-1. In each lane, bands of 70 kDa, expected size of cyclooxygenase-1 in rabbits, can be seen.


    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The extensive phospholipid content and large surface area of the pulmonary vascular bed and airways afford the lung enormous potential for production of eicosanoid metabolites. The lung metabolizes AA via COX, lipoxygenase, and CYP pathways, and products of these enzyme systems are key in modulating physiological responses in the lung (e.g., Ref. 37). CYP AA monooxygenases in the lungs and elsewhere catalyze the formation of epoxyeicosatrienoic acids (EETs) and HETEs (4, 21, 22, 38). Zeldin et al. (40) confirmed CYP2B4/5-catalyzed regio- and stereospecific synthesis of EETs in rabbit lung tissue. The capacity of rabbit lungs to synthesize 20-HETE from AA has been recognized for ~20 years (27). Birks et al. (1) demonstrated that human lung tissue also converts AA to 20-HETE and has CYP4A immunospecific protein. Less information regarding the tissue sources of CYP4A in the lung is available, although these data are clearly important with respect to the potential physiological roles of endogenous CYP4A metabolites. Immunohistochemistry studies identified immunospecific CYP4A protein in nonciliated cells of the proximal airways and a majority of the immunolabeling in rabbit lungs in the end capillary endothelial cells (21). Our data showed conversion of AA to a product that coelutes with authentic 20-HETE and abundant immunospecific protein in rabbit airway tissue (41). The present investigations demonstrate that 19- and 20-HETE are prominent AA metabolites of rabbit airway microsomes. GC-MS analysis confirms that the [14C]AA products that elute with 19- and 20-HETE on RP HPLC are indeed products with the expected mass-to-charge ratios. CYP4A immunospecific protein is evident in airways of all sizes but appears to be more abundant in large compared with small airways (Fig. 5A). Our data are consistent with but significantly extend those of Masters et al. (21), who demonstrated heavy immunohistochemical staining of CYP4A4 in rabbit proximal airways. CYP4A expression does not appear to be confined to the epithelium of the airways because mechanical removal of the epithelium does not diminish immunospecific band density (Fig. 5B). In this regard, CYP4A localization is similar to that of another CYP monooxygenase family, CYP2J (39). CYP2J catalyzes the formation of EETs from AA in lung tissue. Expression of CYP2J isoforms is evident in both nonciliated airway epithelial and bronchial smooth muscle cells of humans and rats (39).

Our data are the first to demonstrate a concentration-dependent relaxation of bronchial rings to 10-8 to 10-6 M 20-HETE. 20-HETE-induced relaxation of bronchi depends on intact epithelium, functional COX activity, and an agent or mechanism of precontraction; 20-HETE-associated relaxation occurred with KCl- and histamine-induced but not with carbachol-induced precontraction. The observation that a COX metabolite of 20-HETE by airway microsomes is a potent bronchodilator lends strength to the hypothesis that the effects of 20-HETE on bronchial tone are indirect and a function of a COX metabolite of 20-HETE. Furthermore, our data underscore the fact that the pharmacological and/or physiological state of the airway profoundly effects the response of the tissue to eicosanoid modulators of tone. Together, these observations raise the possibility that one function of 20-HETE and its metabolite(s) may be modulation of airway tone in rabbits.

With respect to the role of eicosanoids in the control of airway tone, published data are complex and appear to depend on such variables as species (17, 37), route of administration of substrate (31), size of the airway (11), basal tone (33), and other factors such as inspired gas composition and mechanism of preconstriction (e.g., Refs. 5, 7). However, in general, exogenously administered AA causes bronchial constriction (e.g., Refs. 26, 32), and these responses are inhibited by COX and thromboxane synthase inhibitors (8, 34, 35), consistent with bronchoconstrictive activities of thromboxane A2 and PGF2. Our data demonstrating bronchoconstriction with high concentrations of AA are coincident with these reports. Not all COX metabolites are bronchoconstrictive, however. PGE2 at micromolar concentrations relaxed guinea pig bronchial rings (40). Similarly, 5,6- and 8,9-EET cause relaxation of guinea pig bronchial rings (36, 40). 5,6-EET relaxed carbachol- but not histamine- or KCl-contracted guinea pig tracheal rings in an indomethacin-sensitive manner (39). Zeldin et al. (40) reported bronchoconstriction in guinea pig bronchial rings exposed to 20-HETE in concentrations of 10-6 and 10-5 M. Differences between our observations and those of Zeldin et al. (40) may be attributable to utilization of different species or the concentration ranges of 20-HETE tested. We observed that the bronchodilatory effects of 20-HETE were blocked by indomethacin or epithelium removal, strongly suggesting that 20-HETE is an indirect bronchodilator. If a bronchodilatory action of 20-HETE depends on enzymatic conversion to another eicosanoid, then direct bronchoconstrictive effects of 20-HETE might be unmasked at high concentrations when the capacity of COX to convert 20-HETE to its relaxant metabolite was overwhelmed. Our data demonstrating constriction rather than relaxation with micromolar concentrations of 20-HETE in bronchi pretreated with indomethacin (and relaxation when treated with indomethacin vehicle) are consistent with direct constrictive effects of 20-HETE in the absence of metabolism by COX. In addition, species differences in the COX activity or substrate preferences may account for differing responses of rabbit and guinea pig, particularly to high concentrations of 20-HETE.

There is ample precedent for endothelium- or epithelium-localized, COX-dependent effects of 20-HETE on smooth muscle. The vasodilatory actions of 20-HETE in several vascular beds, including splanchnic, coronary, and renal, can be blocked by pretreatment with indomethacin (9, 20, 26). Similarly, Birks et al. (1) demonstrated COX- and concentration-dependent dilation of human pulmonary arteries by 20-HETE. The most straightforward interpretation of these data is that CYP4A metabolites are metabolized by COX into an active vasodilatory compound. There are reports documenting utilization of eicosanoid metabolites (as opposed to AA) as a substrate by COX. Hill et al. (16) showed that COX converts 20-HETE into a variety of products, some of which appear to be unique and many of which are recognized to be vasoactive. We have identified candidate products for this COX metabolite of 20-HETE (see Fig. 3C). One of these products, selected for investigation of biological activity based on a retention time similar to that of a COX product of [14C]AA incubated with the same microsomes and consistent appearance in HPLC studies, elicited relaxation of rabbit bronchi preconstricted with KCl. None of the COX products can be identified based on comigration with known standards in our system (e.g., 6-keto-PGF1alpha ). Thus the chemical nature of these 20-HETE COX metabolite(s) awaits further study.

COX-1 and prostacyclin synthase are ubiquitous in epithelial lung cells of virtually every species studied to date, including humans (e.g., Refs. 6, 14, 33). Rabbit and guinea pig tracheal epithelial cells in vitro express COX and metabolize AA to PGE2 (3, 12). Eicosanoid metabolism of bovine airway epithelial cells in culture can be modified by exposure to ozone, including increased production of prostacyclin, PGE2, PGF2alpha , leukotriene B4, and 5- and 15-HETE (18). Rat pulmonary prostacyclin synthase is expressed late in gestation and can be induced by shear stress, corticosteroids, and antioxidants (14). In addition to respiratory epithelial cells, airway smooth muscle cells express COX-1 and COX-2 and metabolize AA to PGE2 (24). Our data identify COX-1-immunospecific protein as well as COX metabolites of AA and 20-HETE in rabbit airway tissue. Because 20-HETE-induced bronchodilation was not observed in deepithelialized rings, we deduce that the COX responsible for the conversion of 20-HETE to its bronchodilatory product may be localized to the epithelium or that the COX metabolite of 20-HETE must be effecting bronchorelaxation through epithelium-dependent mechanisms. The diminished effect of 20-HETE on epithelium-denuded, precontracted bronchial rings is not a reflection of tissue injury because denuded rings contracted well to KCl, carbachol, and acetylcholine and relaxed on washout of these agents or to exposure to the calcium-channel blocker nifedipine. Furthermore, disruption of epithelial integrity is generally associated with increased rather than decreased sensitivity of bronchial rings to contractile agents (3, 10).

In conclusion, we have demonstrated expression of CYP4A immunospecific protein and 19- and 20-HETE formation in rabbit airway tissue. Submicromolar concentrations of 20-HETE effected a concentration-, epithelial-, and COX-dependent relaxation of KCl-constricted rabbit bronchial rings. 20-HETE-induced bronchorelaxation is not a direct effect of the substance because it depends on intact COX activity. Together, our data suggest that endogenous CYP4A products may serve to modulate airway tone in rabbits. It is tempting to speculate the some forms of human bronchospasm may be related to inadequate production of 20-HETE or dependence on the COX metabolite of 20-HETE to maintain relaxation of the airway (e.g., in patients with triad asthma). Additional studies of the role of these metabolites in modulation of airway tone are needed.


    ACKNOWLEDGEMENTS

We gratefully recognize the contributions of Ying Gao in the preparation and study of bronchial rings, cytochrome P-450 assays, and immunospecific protein studies. We are indebted to Drs. Richard Roman and David Harder for manuscript review, polyclonal cytochrome P-4504A primary antibody, 20-hydroxyeicosatetraenoic acid, and scientific support; to Jayashree Narayanan and Nancy Spitzbarth for scientific support and assistance in completion of the high-performance liquid chromatography studies; and to Kasem Nithipatikom for gas chromatography-mass spectrometry analysis.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-49294 (to E. R. Jacobs) and HL-51055 (to W. B. Campbell) and National Institute of General Medical Sciences Grant GM-31278 (to J. R. Falck).

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. §1734 solely to indicate this fact.

Address for reprint requests: E. R. Jacobs, Cardiovascular Research Center, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226.

Received 26 May 1998; accepted in final form 5 November 1998.


    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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