1 Department of Physiology, 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
bronchi; bronchodilation; eicosanoid; arachidonic acid; cytochrome
P-450
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 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 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
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 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 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
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
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.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
-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
Top
Abstract
Introduction
Methods
Results
Discussion
References
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.
-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.
-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.
0.05 by a two-tailed test
was considered significant.
RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References
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).
View larger version (16K):
[in a new window]
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
108 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).
|
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 (107 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).
|
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 -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).
|
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.
|
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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DISCUSSION |
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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
108 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
106 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-PGF1). 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,
PGF2, 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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