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1 Department of Medicine and 2 Cardiovascular Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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ABSTRACT |
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 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The vasodilatory effect of 20-hydroxyeicosatetraenoic acid (20-HETE) on lung arteries is opposite to the constrictor effect seen in cerebral and renal vessels. These observations raise questions about the cellular localization of 20-HETE-forming isoforms in pulmonary arteries and other tissues. Using in situ hybridization, we demonstrate for the first time CYP4A (a family of cytochrome P-450 enzymes catalyzing formation of 20-HETE from the substrate arachidonic acid) mRNA in pulmonary arterial endothelial and smooth muscle cells, bronchial smooth muscle and bronchial epithelial cells, type I epithelial cells, and macrophages in adult male rat lungs. Moreover, we detect CYP4A protein in rat pulmonary arteries and bronchi as well as cultured endothelial cells. Finally, we identify endogenously formed 20-HETE by using fluorescent HPLC techniques, as well as the capacity to convert arachidonic acid into 20-HETE in pulmonary arteries, bronchi, and endothelium. These data show that 20-HETE is an endogenous product of several pulmonary cell types and is localized to tissues that optimally position it to modulate physiological functions such as smooth muscle tone or electrolyte flux.
20-hydroxyeicosatetraenoic acid; in situ hybridization; vascular tone; vascular smooth muscle; bronchi; cytochrome P-450
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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LOCALIZATION OF cytochrome P-450 4A (CYP4A) in the lung is critical because, unlike in most tissue beds where 20-hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoconstrictor of resistance vessels (9, 15), it dilates microvessels in the lung (1, 17, 18, 48, 49). In addition, dramatic increases in capacity to synthesize 20-HETE during pregnancy in rabbit lungs have been recognized for years (27, 36), and, more recently, its synthesis has been reported in human peripheral lung tissue (1). The extensive phospholipid content and large surface area of the pulmonary vascular bed and airways have therefore recently prompted much interest in the localization of 20-HETE and enzymes that synthesize it in the lung.
Formed by 
-hydroxylation of arachidonic acid (AA), the synthesis of
20-HETE is catalyzed by a number of cytochrome P-450 (CYP450) enzymes of the 4A, 4B, and 4F families (10, 20, 35, 41). Within the group of CYP450 4A, four members, 4A1, 4A2, 4A3,
and 4A8, are known to be expressed in rat tissue (10, 14, 20,
41). CYP4A enzymes are regulated in pathological conditions (22), and the catalytic activity of CYP4A1 to convert AA
to 20-HETE is ~10 times more than that of CYP4A2 or 4A3
(31). There is such a high degree of homology between
members of this family that the only molecular tool available to
distinguish specific members is in vitro reverse transcription followed
by PCRs using short stretches of unique primers (16). Only
recently have the cell types responsible for the formation of 20-HETE
in the lung been investigated in any detail. We have identified the
source of CYP4A-immunospecific protein, mRNA, and 20-HETE production in
peripheral lung tissue of the rabbit (49). The enzymes
were detected in small and large pulmonary arteries, airways, and
smooth muscle cells from small pulmonary arteries showing a wide
distribution of CYP4A in rabbit lung. Semiquantitative comparisons have
revealed greater levels of immunospecific protein in small (external
diameter 400 µm and less) vs. large vessels (49).
We explored the localization and tissue sources of CYP4A and 20-HETE in rat lungs because of availability of better molecular tools for rat enzymes as well as the fact that the only known physiological genomics initiative to correlate genotype with vascular phenotypes is under study using inbred strains of rats (4, 11, 44). In addition, we were interested in examining cross-species pulmonary-specific expression of CYP4A. This report describes endogenous 20-HETE present in airway and vasculature as well as detailed localization of CYP4A1 and -4A2 mRNAs (in situ hybridization studies) and proteins in specific cell types of rat lungs.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Assay for Synthesis of 20-HETE From AA by Airway and Pulmonary Artery Microsomes or Primary Cultures of Bovine Pulmonary Artery Endothelial Cells
The lungs from rats (Sprague-Dawley males, 6 wk old) were harvested under anesthesia. Bronchi and pulmonary arteries were carefully dissected and assiduously separated from surrounding adventitial tissue under a dissecting microscope until only very smooth, glistening, white tissue was visible. Each type of tissue (airways or vessels) was separately pooled and homogenized by use of a handheld tissue homogenizer. Microsomes from whole lung homogenates, isolated cells, or vessels were prepared by differential centrifugation in a modification of methods previously reported (1). Sequential centrifugations at 9,000 g (10 min) and 100,000 g for 1.5 h were performed to generate a microsomal pellet. Protein was quantified according to the method of Bradford (2). Microsomes were separately resuspended in assay buffer (100 mM KH2PO4/K2HPO4, 1 mM EDTA, and 10 mM MgCl2, pH 7.7) and incubated at a protein concentration of 1 mg/ml (200 µl final volume) at 37°C with [1-14C]AA (0.5 µCi/ml; 20 µM), NADPH (1 mM), and a NADPH-regenerating system containing isocitrate and isocitrate dehydrogenase for 30 min (1, 13). In other experiments, first-passage bovine pulmonary artery endothelial cells cultured in 100-mm dishes were washed three times with Hanks' balanced salt solution and incubated for 60 min at 37°C with [1-14C]AA (2 µM) in a buffered saline solution. In all cases, reactions were terminated by acidification with formic acid, and product was extracted with ethyl acetate. The organic phase was back-extracted with distilled water, evaporated under nitrogen, and reconstituted in ethanol. Reaction products were separated on a C18 reverse-phase HPLC column (Supelco, Bellefonte, PA) by using a linear gradient ranging from 100% solvent A (acetonitrile-water-acetic acid 30:70:1) to 100% solvent B (acetonitrile-acetic acid 100:1) over 40 min (17). 14C-labeled products were detected by using a flow-through liquid scintillation cell (HPLC, Beckman System Gold programmable detector module no. 171). Identification of metabolites was based on coelution with authentic standards. The amount of 20-HETE in the sample was calculated by comparing the area of the 20-HETE peak with that of known amount of the substrate, AA, on the same chromatogram.Fluorescent 20-HETE Assays
These assays were performed according to the methods recently published (25, 32). WIT-002 [20-5(Z),14(Z)-hydroxyeicosadienoic acid, Taisho Pharmaceutical, Saitama, Japan] was added as an internal standard to samples, and the lipids from crude tissue homogenates were extracted and labeled with 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate. N,N-disiopropylethylamine was added to catalyze the reaction. The reaction was allowed to proceed for 30 min at room temperature. Labeled AA metabolites were separated on a C18 reverse-phase HPLC column isocratically. The fluorescence was monitored by a fluorescence detector (model L7480; Hitachi, Naperville, IL) at a medium gain sensitivity. The 20-HETE peak was separated from those for dihydroxyeicosatrienoic acids, other HETEs, and epoxyeicosatrienoic acids (EETs) and quantitated by comparing the area of the 20-HETE peak with that of the internal standard.Nonradioactive In Situ Hybridization
PCR of CYP4A sequence. CYP4A cDNA was amplified from plasmid carrying full-length cDNA (30, 42, 46) by using CYP4A1 and -4A2 primers (16). PCR was carried out with a hot start and 30 cycles of denaturation (94°C), annealing (55°C), and elongation (72°C). Amplified PCR products were resolved on a 1.5% low-melting agarose gel, stained with ethidium bromide for size verification, excised from the gel, and purified with the Qiaex II gel extraction kit (Qiagen, Valencia, CA).
Subcloning and sequencing of CYP4A products. The isolated CYP4A cDNAs were subcloned into the EcoRI site of plasmid pCR II (TA cloning kit, Invitrogen, Carlsbad, CA), between the SP6 and T7 promoters. The plasmids carry the lacZ gene for blue/white color to identify presence of inserts and the ampicillin resistance gene for selection on growth media. The plasmids with inserts were amplified and purified by column chromatography, using the Qiagen plasmid purification system according to the manufacturer's instructions (Qiagen, Valencia CA). Purified plasmids were sequenced to determine the orientation and confirm correspondence to CYP4A1 and 4A2 cDNA. DNA sequencing was carried out by the Shared Protein and Nucleic Acid Laboratory at the Medical College of Wisconsin, Milwaukee, with the use of an ABI PRISM 301 genetic analyzer.
Riboprobe synthesis. Antisense and sense riboprobes were generated after sequencing by in vitro transcription using sets of restriction enzymes (BamHI or EcoRV) and RNA polymerases (T7 or Sp6). Riboprobes were labeled with digoxigenin-11-UTP (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer's instructions.
Cryostat sectioning and fixing. The vascular beds of the lungs were perfused by injection of 15 ml RNase-free cold phosphate-buffered saline (PBS; 140 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4, pH 7.4) into the right ventricle of an anesthetized rat, after which the lung was removed. Frozen 8-µm lung sections were cut with a cryostat, mounted on slides, and fixed with 4% paraformaldehyde followed by washes for 5 min in 2× SSC (1× SSC contains 150 mM NaCl, and 15 mM sodium citrate, pH 7.0).
Hybridization. The sections were placed in a humidified chamber; covered for 30 min at 50°C with 50 µl prehybridization solution containing 50% formamide, 5× SSC, 5× Denhardt's reagent, and 100 µg/ml denatured, sheared herring sperm DNA; and then probed with the labeled riboprobe (30 ng/ml) for 12 h at 60°C. The sections were washed three times with 2× SSC at room temperature and incubated in 10 mM Tris buffer (pH 8.0) containing 0.5 M NaCl and 30 U/ml RNase A to destroy RNA-DNA hybrids and single-strand RNA, for 60 min at 37°C. The sections were rinsed with 2× SSC, 1× SSC, and 0.5× SSC sequentially for 5 min at room temperature and then washed with 0.1× SSC for 60 min at 65°C. The sections were washed with 0.1 M phosphate buffer (PB), pH 7.4, and were blocked in carrageen mix containing 0.25% carrageen, 0.3% Triton X-100 in 0.1 M PB for 2 h at room temperature, followed by incubation with a 1:2,000 dilution of anti-dig-AP (alkaline phosphatase conjugate MAb, Boehringer Mannheim) in carrageenan mix. The sections were washed two times with 0.1 M PB containing 0.01% thimerosal, two times with TBS (100 mM Tris buffer and 150 mM NaCl, pH 7.5) for 10 min, and one time with alkaline salt buffer (ASB) (100 mM Tris buffer, 150 mM NaCl, 50 mM MgCl2, pH 9.5) for 5 min. Color reaction was carried out in the dark for 2-24 h. Immunoreactivity was visualized by incubation of sections with the alkaline phosphatase substrate 5-bromo-4-chloro-3-indoyl-phosphate with nitro blue tetrazolium. These sections were viewed and photographed with a microscope equipped for both light and epifluorescence (Eclips 600, Nikon) with an attached digital camera.
Staining of Type I and Type II Cells and Macrophages
After in situ hybridization studies, some slides were processed to identify type I and II cells and macrophages by double staining. They were blocked with 5% BSA containing 0.1% Triton X-100 for 30 min at room temperature. Slides were then incubated with Texas red-coupled Maclura pomifera lectin (MPA; Sigma Chemical, catalog no. L 2013), which labels type II cells and alveolar macrophages (19), and FITC-coupled Bachhinia purpura lectin (BPL; Sigma Chemical, catalog no. L 9512) to label type I epithelial cells (19). Slides were rinsed twice in PBS, then finally in tap water, mounted with a coverslip, and visualized with a Nikon microscope equipped with epifluorescence (Eclips 600) and image-acquisition hardware and software.Immunohistochemical Staining of Endothelial Cells
Lungs were flushed, fixed, and sectioned as described above. After blocking overnight with 5% BSA in PBS with 0.1% Triton X-100, they were incubated with a primary antibody against rat platelet endothelial cell adhesion molecule-1 (PECAM-1; Ref. 39; generous gift from Dr. Peter Newman, Blood Center of Southeastern Wisconsin, Milwaukee, WI) at a dilution of 1:50 vol/vol in 5% BSA overnight at 4°C. After washing, the slides were exposed to a secondary antibody (goat anti-rabbit IgG conjugated with FITC, Molecular Probes no. A-11008) and visualized as per lectins above.Endothelial Cell Culture
Rat or bovine endothelial cells were prepared from pulmonary arteries harvested from two to three rats or a lobe of bovine lung obtained from a local slaughterhouse. Pulmonary arteries were slit open along their lengths and washed with PBS to remove blood (29). The vessels were cut into ~3- to 5-mm2 pieces with a sharp scissors and placed with the lumen side down onto a 100-mm tissue culture dish for 5 min. After they adhered, they were covered with medium (RPMI containing 20% FBS) and allowed to grow for 3 days in a tissue culture incubator. Tissue pieces were then lifted out of the medium, and adherent endothelial cells were allowed to grow. After several days, trypsinized cells were diluted and plated in a 96-well dish with 1-2 cells/well to select for a pure population of cells on the basis of characteristic cobblestone appearance. Selected cell populations were expanded, cultured for Western analysis, and probed with the endothelial cell marker PECAM for verification of population purity. Bovine pulmonary artery endothelial cells (PAECs) were studied without subculturing from 96-well plates. Rat and bovine PAECs were ~75% and 90% PECAM positive, respectively (n = 6 each). First through fourth passage cells were used for studies detailed in this work.Immunospecific Identification of CYP4A Protein
Microsomal suspensions prepared from cultured endothelial cells as well as pulmonary arteries were separated by electrophoresis on a 10% denaturing sodium dodecyl sulfate-polyacrylamide gel and transferred to a nitrocellulose membrane (49). Nonspecific binding was blocked by incubating the membrane in TBS in 10% (CYP450 4A) nonfat milk overnight, followed by three washes with TBS. The nitrocellulose membrane was incubated for 2 h at room temperature with a primary antibody (polyclonal anti-4A1 and -4A3 antibody from Gentest, Woburn, MA). The membrane was rinsed three times before incubation with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:1,000) and then visualized by use of enhanced chemiluminescence. X-ray film was developed on the Kodak XOMAT developer.| |
RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Conversion of AA Into 20-HETE in Microsomes From Bronchi and Pulmonary Arteries and Primary Cultures of Bovine PAECs
Microsomes from bronchi and pulmonary arteries or bovine PAECs were incubated with radiolabeled substrate [1-14C]AA and cofactors for 30 min at 37°C (see METHODS). Lipid products of these reactions were resolved by reverse-phase HPLC and preliminarily identified on the basis of coelution with authentic standards as shown in the chromatograms in Fig. 1. In samples from all three sources, we observed clear peaks at ~23 min coeluting with our standard 20-HETE (from pregnant rabbit lung, not shown) whereas the excess substrate (AA) eluted at 34 min.
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Endogenously Formed 20-HETE in Pulmonary Arteries and Bronchi
Pulmonary arteries and bronchi were also analyzed for the presence of endogenous 20-HETE by a sensitive fluorescent assay. Fluorescently tagged cellular lipids were resolved by HPLC (25), and the eluted fractions were scanned by a fluorescent detector to identify retention times of labeled peaks, which were compared with a known amount of an internal standard (WIT-002) that was included to quantitate the 20-HETE peak in each sample. A comparison of the results from airways vs. pulmonary arteries is shown graphically in Fig. 2. The 20-HETE content from these two sources ranged from 200 to 300 pg/g of tissue.
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Distribution of CYP4A1 mRNA in the Lung
To precisely localize expression of one of the major enzyme families that catalyze conversion of AA to 20-HETE, we probed frozen sections of rat lung with riboprobes derived from the coding sequences of CYP4A1 and -4A2 cDNA, which were subcloned in the plasmid pCR II. The inserted fragments were confirmed to be CYP4A1 and -4A2 by DNA sequencing, and probes were prepared from them as described in METHODS. In situ hybridization with these probes revealed the presence of CYP4A-specific signal in a number of cell types as shown in Fig. 3, A-L.
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Bronchi. Intense staining within the smooth muscle in the bronchi was apparent with the CYP4A1 and CYP4A2 antisense probes, which were used to detect complementary CYP4A mRNA sequence. The results suggest that the main source of CYP4A in the airways is the smooth muscle. Interestingly, specific staining was also detected in the epithelial lining of the airways. This is readily observed under higher magnification as seen in Fig. 3B (see arrows). Negative controls of hybridization with the sense probes for the CYP4A1 and CYP2 sequences are shown in Fig. 3, M and N. The overall background intensity was low, and the images had minimal edge-effect staining, in contrast to the specific signal with the CYP4A antisense probes.
Vasculature. The most prominent areas of staining within the lung vasculature were in the smooth muscles of medium- and large-size arteries. Figure 3, C and D, shows dark staining in multilayers of muscle cells within the walls of the arteries. These vessels range from ~3 to 4 mm in diameter and represent larger branches of pulmonary arteries of the rat. Negative controls (sense probes) showed very faint and diffuse staining when observed under identical microscopic settings (Fig. 3, O and P). Inspection with higher magnification (Fig. 3D) revealed the presence of specific staining in endothelial cells of medium and small pulmonary arteries. CYP4A expression has not been reported previously in endothelial cells from any tissue beds other than lungs of pregnant female rabbits and was therefore examined more closely. Care was taken not to damage the endothelial linings during perfusion of the lung. Smaller vessels in the section that were cut at angles favoring exposure of the endothelium showed very clear staining of endothelial cells, as marked by arrows in Fig. 3E. The sections were counterstained with antibody for the endothelial-specific marker PECAM-1 (Fig. 3F) to confirm the identity of the cells. The results were also confirmed by carrying out Western analysis with cultured endothelial cells (see Western Analysis of Rat Lung Tissue With CYP4A Antibody). Thus CYP4A is expressed in both vascular smooth muscle and endothelial cells of rat pulmonary arteries.
Peripheral Lung
Examination of rat lung sections with the CYP4A1 and 4A2 antisense riboprobes after in situ hybridization showed dense staining in flattened parenchymal cells of the alveoli. Sections were counterstained with BPL and MPA lectins (see METHODS), which bind to type I and type II pneumocytes, respectively. Alveolar macrophages are also stained by MPA (Fig. 3H). These data demonstrate that type I pneumocytes frequently and type II pneumocytes rarely express CYP4A specific mRNAs (Fig. 3, I-L). In addition, interstitial and alveolar macrophages also contained CYP4A specific signal (Fig. 3, G and H).Western Analysis of Rat Lung Tissue With CYP4A Antibody
To confirm expression of CYP4A protein in endothelial cells, Western analysis with antibody raised against rat CYP4A protein was performed. Microsomes from rat pulmonary arteries (Fig. 4, bottom) as well as lysates of rat and bovine endothelial cells (Fig. 4, top) were examined for presence of CYP4A protein, and immunospecific bands were observed at the expected molecular size (~55 kDa) in all the samples, as seen in Fig. 4.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Very little is known regarding the location of CYP4A in the lung of any species, but these data are critically important in that sources of 20-HETE production have a profound effect on functional implications. Immunohistochemical studies identified CYP4A protein in nonciliated cells of the proximal airways of pregnant and nonpregnant rabbits, whereas endothelial cells of end capillaries accounted for most of the pulmonary expression during pregnancy (26). In this study, we demonstrate for the first time CYP4A mRNA in pulmonary vascular endothelial and smooth muscle cells, bronchial smooth muscle and epithelial cells, type I epithelial cells, and macrophages of male rat lungs. Moreover, we find CYP4A protein in rat pulmonary arteries and bronchi as well as low-passage endothelial cells in culture. Finally, our data identify endogenously formed 20-HETE as well as the capacity to convert AA into 20-HETE in pulmonary arteries and bronchi. Colocalization of CYP4A mRNA, protein, and product in these cells provides the strongest data to date that 20-HETE is an endogenous product of several pulmonary cell types and is localized to tissues that optimally position it to modulate pulmonary vascular and/or airway tone.
Perhaps the most novel aspect of pulmonary CYP4A expression is localization to endothelial cells. Human endothelial cells express enzymes of the CYP2C8, -2C9 (7), -2J2 (33), -3A, and -2B1 families that all produce EETs. In addition, previous studies have verified expression of 20-HETE-forming CYP4A1, -4A2, and -4A3 isoforms (or homologs of the same) in a variety of tissues and species, including vascular smooth muscle cells. Message for the CYP4A2 and -4A3 isoforms is found in small arterioles in the kidney (15), brain (9), and skeletal muscle beds (8, 23) of rats. Moreover, CYP4A expression in vascular smooth muscle cells of the cerebral and renal circulations has been clearly established by immunohistochemistry and product formation. However, the only other evidence of endothelial cell CYP4A expression is based on immunohistochemical studies demonstrating high levels of CYP4A in end-capillary endothelial cells of pregnant rabbits (26). The signal in the endothelium of nonpregnant rabbits may have been either not present or too low to be detected by the antibody used, or endogenous mRNA may not have been getting translated into protein. The affinity of the antigen-antibody reaction, which is variable, determines the sensitivity of the immunohistochemical assay. Detection by in situ hybridization on the basis of RNA-RNA annealing depends on controlled physical factors such as salt concentration and temperature so that specific signal reflects the presence of mRNA. In situ localization of signal in the endothelium was confirmed by double immunostaining with the endothelial cell-specific marker, PECAM-1. Primary cultures of bovine PAECs convert AA into products with an elution time identical to that of authentic 20-HETE. To ascertain whether CYP4A protein was also present in the endothelial cells, a Western blot using anti-rat CYP4A antibody was performed. Microsomes from rat pulmonary arteries as well as lysates of cultured endothelial cells from rat and bovine pulmonary arteries showed presence of CYP4A protein at the expected molecular mass of ~55 kDa (Fig. 4). We therefore feel confident that both mRNA and protein for CYP4A are expressed in endothelial cells in rat lungs. Thus our data confirm pulmonary artery vascular smooth muscle cell expression, as in systemic vascular beds but define a unique CYP4A localization in PAECs.
Microsomal conversion of AA to 20-HETE and CYP4A protein in male rat lungs generally follows the distribution in nonpregnant rabbit lungs (49). Rat airways, pulmonary arteries, and peripheral lung tissues express CYP4A protein and synthesize 20-HETE. Other reports of lung microsomal metabolism of AA to C-20 alcohols have been reported in rabbit as well as guinea pig lungs (21, 34). In addition and more importantly, our data are the first to demonstrate that this reaction must be occurring naturally in the lung, because endogenous 20-HETE was detected in both the airways and pulmonary arteries (Fig. 2). The amount of 20-HETE we detected is in the same range as that reported for each isomer of EET in the rabbit lung (47). The most concentrated isomer of EET, 14,15-EET, was present at 147 ng/g lung (47), whereas the 20-HETE was at 200-300 ng/g lung.
Colocalization of CYP4A message, protein, and product do not inevitably
link expression and product formation. In addition to CYP4A isoforms,
enzymes of the CYP4B and 4F families also catalyze -hydroxylase
conversions of AA into 20-HETE (37). Evidence for the
formation of 20-HETE from [14C]AA substrate by canine
polymorphonuclear leukocytes has been reported (38),
although the participating CYP450 -oxidating enzymes were not
identified in this study. Thus although our data demonstrate message,
protein, and lipid products in particular pulmonary cell types, we
cannot exclude the possibility that other CYP isoforms may contribute
to 20-HETE formation in the lung.
There is good precedent for expression of 20-HETE forming CYP isoforms
in epithelial cells outside the lung. CYP4A is highly expressed in rat
renal epithelial cells, including the thick ascending loop of Henley
(TALH) and proximal tubular epithelial cells (28). 20-HETE
appears to play a major role in the regulation of chloride transport in
the TALH by inhibiting
Na+-K+-2Cl
cotransport in this
nephron segment (3, 6). In renal tubular epithelial cells,
20-HETE inhibits Na+-K+-ATPase activity and
also sodium transport in isolated perfused rabbit proximal tubules
(40). We have no information regarding the functional
implications of CYP4A expression in pulmonary epithelium, other than
observations that 20-HETE appears to modulate the tone of bronchial
rings (17, 18, 47). However, our observations of CYP4A in
bronchial epithelial and type I pulmonary epithelial cells of the lung
suggest that 20-HETE-forming isoforms might be well positioned to
control fluid and electrolyte flux in the airways.
The implications of the expression pattern of localization of CYP4A1 and -4A2 in rat pulmonary arteries center on the potential role of 20-HETE in regulating vascular tone. 20-HETE is a constrictor of renal (24, 45), cerebral (9, 13), and skeletal muscle arterioles (8, 23). It is more potent in smaller arterioles (<100 µm) than larger vessels such as the aorta (37). Interestingly, 20-HETE has been seen to behave as an endothelial-dependent vasodilator in human (1) as well as rabbit (49) lungs. The upregulation of 20-HETE during pregnancy may be beneficial in reducing pulmonary vascular tone in the face of increased blood volume and cardiac output, especially that experienced during parturition. Induction of the 20-HETE-synthesizing system therefore has the potential to negate the effects of conditions such as preeclampsia, although such a role remains entirely speculative at this point.
In summary, widespread distribution of CYP4A in the lung demonstrated in this work suggests a host of possible physiological and/or pathophysiological functions and raises the possibility that CYP4A expression in one cell type (e.g., epithelium) may have distinct functional implications from expression in another cell type (such as alveolar macrophages or endothelial cells). These observations set the stage for investigations to determine the functional implications of cyclooxygenase CYP4A in discrete pulmonary cell types, as well as the effect of stresses such as hypoxia, pregnancy, cyclooxygenase inhibition, and others on expression in these tissues.
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ACKNOWLEDGEMENTS |
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Endogenous 20-HETE assays were developed and done by Kristopher Maier and Richard Roman in the Department of Physiology, Medical College of Wisconsin. We acknowledge Professor Richard Roman and Michael Aebly for providing the primers for the PCR reactions. We thank Ying Gao and Ryan P. McAndrew for invaluable technical assistance.
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FOOTNOTES |
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Financial aid was from National Heart, Lung, and Blood Institute Grants RO1 HL-49294 and PO1 HL-59996.
Address for reprint requests and other correspondence: E. R. Jacobs, Cardiovascular Center, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: ejacobs{at}mcw.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.
First published January 18, 2002;10.1152/japplphysiol.01159.2001
Received 26 November 2001; accepted in final form 11 January 2002.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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