We previously reported that the levels of epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) are depressed in microsomes prepared from lungs of rats with acute Pseudomonas pneumonia. We also showed a potential role for cytochrome P-450 (CYP) metabolites of arachidonic acid (AA) in contractile responses of both normal pulmonary arteries and pulmonary arteries from rats with pneumonia. The CYP2J subfamily enzymes (endogenous source of EETs and HETEs) are constitutively expressed in human and rat lungs where they are localized in vascular smooth muscle and endothelium. The purpose of this study was to determine if CYP2J proteins are modified in pneumonia. Pseudomonas organisms were injected via a tracheostomy in the lungs of rats. Later (44 h), lungs were frozen, and microsomes were prepared from pneumonia and control rat lung homogenates. Lung microsomal proteins were then immunoblotted with anti-CYP2B1/2B2, anti-CYP4A, anti-CYP2J9pep2 (which reacts with rat CYP2J3), anti-CYP2J6pep1 (which reacts with rat CYP2J4), anti-CYP2J2pep4, or anti-CYP2J2pep3 (both of which react with all known CYP2J isozymes). Western blotting revealed a prominent 55-kDa band with anti-CYP2J2pep3, anti-CYP2J2pep4, and anti-CYP2J6pep1 (but not anti-CYP2J9pep2) that was reduced in pneumonia compared with control lung microsomes. The CYP2B bands (51-52 kDa) were less prominent and not different between pneumonia and control lungs. CYP4A proteins (20-HETE sources) were not detected in rat lung microsomes. Therefore, rat lung contains a protein with immunological characteristics similar to CYP2J4, and this CYP is reduced after pneumonia. We speculate that CYP2J (but not CYP2B) enzymes and their AA metabolic products (EETs) are involved in the modulation of pulmonary vascular tone in pneumonia in rats.
- cytochrome P-450
- Western blotting
- cytochrome P-2J4 and cytochrome P-4A protein content
- peptide antibodies
there is considerable evidence in the literature implicating the cytochrome P-450 (CYP) metabolites of arachidonic acid (AA), 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs), as possible intracellular signaling molecules that play a role in constrictor or dilator vascular responses (34, 38, 40). In addition, CYP products, especially EETs, are released from isolated and perfused human lungs after an inflammatory challenge (17). We have previously demonstrated that the rate of production of CYP metabolites of AA, 20-HETE and EETs, is depressed in lung microsomes from rats with pneumonia compared with control rats (49). In addition, we showed that 20-HETE and EETs are potent constrictors of small pulmonary arteries from rats. However, contractility to 20-HETE and EETs is depressed in pulmonary arteries isolated from rats with acute Pseudomonas pneumonia (49). This indicates a potential role for these metabolites in contractile responses of both normal pulmonary blood vessels and in the setting of pneumonia.
In hepatic tissues, evidence of a decline in CYP protein content in models of inflammation and infection is available (27, 36, 37, 39). However, in our model of pulmonary infection, we did not know whether the decline in CYP metabolites in lungs of rats with acute pneumonia is because of inhibition of activity of CYP enzymes by inflammatory mediators or is a result of a decline in protein levels of the enzymes.
Evidence from the literature indicates that isozymes of the CYP2J subfamily, which convert AA to EETs and HETEs, are constitutively expressed in human and rat lungs. CYP2J is known to be localized in vascular smooth muscle and endothelium of lung (51). Another possible source of EETs in rodent lungs is CYP2B (18, 32, 52). Yet, whether or not these enzymes are modulated by Pseudomonas pneumonia infection in lung is unknown. Therefore, the aim of this study was to determine if CYP2J or CYP2B proteins, known AA epoxygenases, are altered in lungs isolated from a rat model of acute Pseudomonas pneumonia.
Recent studies, aimed at determining the source of 20-HETE and the location of CYP4A in the lungs, demonstrated that small pulmonary arteries from rabbit lungs express CYP4A proteins and that vascular smooth muscle cells derived from these arteries synthesize 20-HETE (54). Similarly, microsomes from rat pulmonary arteries and microsomes of cultured endothelial cells from rat and bovine pulmonary arteries showed the presence of CYP4A protein (55). Therefore, we investigated if CYP4A protein, a known source of 20-HETE, is altered in lung microsomes from rats with pneumonia.
In this study, we did not detect significant amounts of CYP4A protein in lung microsomes from control and pneumonia rats. However, using peptide-based antibodies to members of the CYP2J subfamily, we identified in rat lung a protein with immunological characteristics similar to CYP2J4. Furthermore, we showed that this CYP isozyme is reduced in the setting of pneumonia, indicating a possible role for CYP2J enzymes and their products in the response to this disease.
MATERIALS AND METHODS
All animals used in this study were cared for following the principles and guidelines of the Canadian Council on Animal Care and were supervised by a veterinarian. In addition, the ethics review committee at the University of Western Ontario (London, ON, Canada) approved all protocols.
Acute pneumonia model. The acute pneumonia rats were prepared as described previously (46). Briefly, male Sprague-Dawley rats (275-350 g) were randomized to a pneumonia group or control group. Animals in both groups were anaesthetized with halothane, and a jugular venous line was placed for fluid administration. Animals in the pneumonia group were injected intratracheally with 0.15 ml saline containing 3 × 108 colony-forming units/ml through a tracheostomy. Within 36 h, this instillation of bacteria produced an acute localized pneumonia in the left lung, with the right lung appearing grossly normal. Animals in the control group had a tracheostomy only. Postoperatively, the rats were housed separately and allowed free access to standard rat chow and water. Fluid was maintained, for all rats, by a continuous infusion of heparinized saline (1 U/ml) at 2 ml/h. Fentanyl (1.0 μg/ml) was added to the venous infusion for analgesia. After surgery (44 h), rats were anesthetized with pentobarbital sodium (30 mg/kg iv), and the thorax was opened. The heart and lungs were removed en bloc and perfused through the pulmonary artery with Krebs bicarbonate solution (in mM: 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4·7H2O, 1.2 KH2PO4, 11.1 dextrose, and 22.1 NaHCO3, pH = 7.4). Lungs were placed in labeled cryogenic vials, sealed, and frozen immediately in liquid nitrogen. All vials were stored at -80°C until used.
Preparation of lung microsomes. Microsomal fractions from control and pneumonia lung homogenates were prepared by established methods as previously described (2, 49). Microsomal fractions from naive lungs were also prepared. Naive lungs were obtained from untreated rats from the same batch of animals randomized to control or pneumonia groups. Microsomes were divided into 200-μl aliquots and frozen immediately until needed for Western blotting. A 50-μl aliquot of the microsomes was used for protein determination by the method of Lowry et al. (21) using BSA as the standard.
Preparation of antibodies. The recombinant CYP2J2, CYP2J3, CYP2J5, CYP2J6, CYP2J8, and CYP2J9 protein standards were prepared as previously described (22, 23, 30, 43, 44). Recombinant CYP2J4 was a generous gift from Dr. Laurence Kaminsky (Wadsworth Center, New York State Department of Health). Polyclonal antibodies against the mouse CYP2J9 polypeptide GQARQPNLADRD (anti-CYP2J9pep2) were raised in New Zealand White rabbits as previously described (30). This antibody has been shown to immunoreact with mouse CYP2J9 and rat CYP2J3 but does not cross-react with other CYP2J isoforms (30). Polyclonal antibodies against the mouse CYP2J6 polypeptide QMEQNIMNRPLSVMQ (anti-CYP2J6pep1) were similarly prepared in rabbits. This antibody immunoreacts with mouse CYP2J6 and rat CYP2J4 but does not cross-react with other CYP2J isoforms. Polyclonal antibodies against the human CYP2J2 polypeptides RESMPYTNAVIHEVQRMGNIIPQN (anti-CYP2J2pep3) and FNPDHFLENGQFKKRE (anti-CYP2J2pep4) were raised in rabbits and cross-react with all known CYP2J subfamily P-450s. None of these antibodies is known to cross-react with non-CYP2J subfamily P-450s. Preimmune serum, collected from the rabbits before immunizations, did not cross-react with recombinant CYP2J isozymes.
SDS-PAGE and Western blotting for CYP proteins. Lung microsomal proteins were electrophoresed on SDS-10% polyacrylamide gels, and the resolved proteins were transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes (Amersham Canada, Oakville, ON, Canada). Membranes were immunoblotted using anti-CYP2B1/2B2 (dilution: 1:100; Cedarlane Laboratories, Hornby, ON, Canada), anti-CYP2J9pep2, anti-CYP2J6pep1, anti-CYP2J2pep4, or anti-CYP2J2pep3. All the CYP2J antibodies were used at a dilution of 1:1,000. Bound antibodies were detected using goat anti-rabbit IgG conjugated with horseradish peroxidase (dilution: 1:2,000; Promega, Madison, WI) and the enhanced chemiluminescence Western blotting detection system (ECL Plus; Amersham Canada). Relative molecular weights of proteins were determined by running a prestained protein marker (Promega) on all gels. In addition, microsomes from Sf9 cells expressing recombinant CYP2J3 were used as a standard. Films were scanned, and the intensities of the bands were quantified via photodensitometry. Multianalyst 1.1 (Bio-Rad Laboratories, Hercules, CA) was used for measuring optical densities (OD). Band densities were expressed as adjustable volumes (OD × mm2).
Stripping and reprobing with multiple antibodies. Membranes were incubated in a hybridization incubator at 50°C in stripping buffer (see below) with gentle shaking for 2 h. Membranes were then washed in Tris-buffered saline (TBS) with 0.1% Tween 20, dipped in polyvinyl alcohol (1 mg/500 ml TBS blocking buffer, stored at 4°C), and blocked for 2 h in Blocker Blotto in TBS (Pierce, Rockford, IL). Western blotting was continued beginning with primary antibody. Stripping buffer composition (100 ml) was as follows: 6.25 ml of 1 M Tris (pH 6.7), 20 m1 of 10% SDS, 0.69 m1 of 2-mercaptoethanol (14.4 M), and 73.06 ml distilled water.
CYP4A. A similar method as above was followed for Western blotting. PVDF membranes were immunoblotted with anti-rat CYP4A polyclonal antibody (Chemicon International, Temecula, CA) at 1:1,500 dilution. Rat kidney microsomes were used as a positive control for CYP4A isozymes.
Chemicals. Reagents needed for Krebs solution were purchased from Sigma (Mississauga, ON, Canada). Other reagents needed for SDS-PAGE electrophoresis, transfer, and Western blotting of proteins were purchased from Amersham Canada. Blotto in TBS (Pierce) was used for blocking the PVDF membranes.
Data analysis. Densitometry results are expressed as means ± SE of n values, where n is the number of rats. Results were compared using ANOVA followed by Bonferroni's posttests. A value of P < 0.05 was considered significant.
Cross-reactivities of antibodies. Blots in Fig. 1 demonstrate the cross-reactivity of the peptide antibodies (used in this study) with recombinant CYP2J isoforms. As shown, anti-CYP2J2pep3, anti-CYP2J2pep4, and anti-CYP2J6pep1 immunoreact with recombinant CYP2J4, but anti-CYP2J9pep2 does not.
Pulmonary microsomal CYP proteins. Western blotting revealed a prominent 55-kDa band with anti-CYP2J2pep3, anti-CYP2J2pep4, and anti-CYP2J6pep1 (but not anti-CYP2J9pep2) that was reduced in pneumonia lung compared with control lung microsomes (Fig. 2). Figure 2, A and B, identifies bands in the 50- to 60-kDa range that are CYP2Js based on reactivity with the various antibodies (Fig. 1). Anti-CYP2J6pep1 indicates the 55-kDa band that identifies CYP2J4 in rat lung (Fig. 2). The intensity of the 55-kDa bands in naive lung microsomes was not significantly different from that in control lungs when equal amounts of microsomal protein (20 μg) were loaded, as demonstrated in Fig. 2C.
Stripping and reprobing the same membranes with anti-CYP2B1/2 showed that the intensity of the CYP2B bands at 51-52 kDa were less prominent and not different between pneumonia and control lungs, as shown in Fig. 2B.
Data from lung microsomes of naive, control, and pneumonia rats compared with CYP2J3 are normalized and presented as a fraction of control [= adjustable volume OD (OD × mm2)/highest adjustable volume (OD × mm2) measured in each blot] (Fig. 2C).
Antibody for CYP4A was also used to determine if these proteins could be identified in rat lung and if their concentration was modified in Pseudomonas pneumonia lungs. CYP4A proteins were not detected in lung microsomes from pneumonia or control rats compared with rat kidney microsomes used as positive controls (Fig. 3).
Pulmonary CYP2J4 in pneumonia. To make sure protein and antibody concentrations were in the range that could be used for comparison between pneumonia and control lung microsomes, Western blots with increasing protein concentrations of control (Fig. 4A) or pneumonia (Fig. 4B) microsomes were also performed. Western blotting of control lung microsomes revealed the 55-kDa band with anti-CYP2J2pep3, anti-CYP2J2pep4, and anti-CYP2J6pep1 (but not anti-CYP2J9pep2) showing augmented band density with increased protein loading of control lung microsomes (Fig. 4A). In comparison, the 55-kDa band revealed with anti-CYP2J2pep3, anti-CYP2J2pep4, and anti-CYP2J6pep1 (but not anti-CYP2J9pep2) was less prominent in pneumonia lung microsomes, even with increased protein loading (Fig. 4B). Data from Western blots of control and pneumonia lung microsomes (Fig. 4) were pooled and expressed as a fraction of control [adjustable volume optical density (OD × mm2)/highest adjustable volume (OD × mm2) measured in each blot] for comparison (Fig. 5). These data indicated that the reduced CYP2J band in pneumonia was definitely the result of a reduction of CYP2J4 protein. The fact that the decreased content of CYP2J4 in pneumonia lungs might be because of edema and influx of exogenous proteins was ruled out, since there was no attenuation of the CYP2B1/2 bands in pneumonia compared with control lung microsomes (Fig. 2, B and C).
In acute lung injury (such as occurs in pneumonia or systemic sepsis), attenuated pulmonary artery contractility has been demonstrated both in vivo (8, 10, 11) and in vitro (24, 42, 47, 49) to a number of stimuli, including hypoxia, ANG II, norepinephrine, potassium chloride, 20-HETE, and EETs. Specifically, depressed contractility of small pulmonary artery rings dissected from lungs of rats with chronic (47) or acute (49) Pseudomonas pneumonia or sepsis secondary to peritonitis (24, 48) has been demonstrated. Furthermore, sepsis and pneumonia impair hypoxic pulmonary vasoconstriction, which occurs because of contraction of smooth muscle in the walls of the small arterioles in the hypoxic regions of lung, thereby reducing arterial oxygenation and enhancing hypoxemia in animal models (10, 11, 41) and in humans (1, 6, 19). The mechanism of this loss of contractility in response to environmental and pharmacological stimuli is not fully understood.
We previously demonstrated that acute Pseudomonas aeruginosa pneumonia caused depressed contractility of intrapulmonary arteries of rats to a number of contractile agonists (phenylephrine, potassium chloride, PGF2α, EETs, and 20-HETE) and that factors other than nitric oxide and dilator cyclooxygenase metabolites could contribute to the depressed pulmonary artery contractility observed in pneumonia (46, 49). In addition, Liu et al. (20) demonstrated in isolated perfused lungs of rats that inhibition of either nitric oxide synthase or cyclooxygenase caused little rise in pulmonary artery pressure, whereas inhibition of both enzymes caused marked increases in pulmonary artery pressure compared with either enzyme inhibited alone (20). This observation supported our conclusions that the depressed pulmonary artery contractility is correlated with the reduced rate of formation of constrictor CYP metabolites (EETs and 20-HETE) in lung microsomes from pneumonia compared with control rats (49). Therefore, a role for these CYP metabolites of AA in the contractile responses of small pulmonary arteries either as direct-acting or as intracellular signaling molecules to other agonists is implicated.
In this study, Western blotting of rat microsomal protein with the CYP2J antibodies revealed a prominent band for CYP2J4 at ∼55 kDa that was reduced in pneumonia lungs compared with control lungs. In addition, the CYP2B bands at ∼51-52 kDa were less prominent compared with the CYP2J bands and were not different between pneumonia and control lungs. This is the first study to demonstrate that CYP2J4 (not CYP2J3) is the most abundant CYP2J isoform in rat lung under basal conditions and that this CYP is reduced by inflammation resulting from infection.
CYP metabolites of AA are produced by lung microsomes of different species and exist endogenously in lung tissue (16). Whether epoxygenation products or ω-hydroxylation products are formed depends on the specific CYP isoform. Thus CYP4A proteins have been indicated as a source of 20-HETE in rabbit, rat, and human lung microsomes (3, 54, 55), whereas CYP2J and CYP2B produce mainly EETs and omega HETEs (18, 51, 52). Prominent expression of CYP2J in vascular smooth muscle and vascular endothelium has been demonstrated in human and rat lung (51). CYP2J1 has been identified in rabbit, CYP2J2 in human, CYP2J3 and CYP2J4 in rat, and CYP2J5 and CYP2J6 in mouse tissues (35). In lung microsomes from our control and pneumonia rats, we did not detect significant amounts of CYP4A protein by SDS-PAGE and Western blotting. However, we found a band of CYP2B protein that was not modified in pneumonia lung microsomes and a more prominent CYP2J band that was reduced in pneumonia lung microsomes. Prior studies with rat lung microsomes used antibodies that recognized both CYP2J3 and CYP2J4 isoforms and were not able to identify which of the two CYP2J isozymes was more concentrated in rat lung (51). With the use of recombinant CYP2J4 and the newer antibodies, the data presented here clearly show that CYP2J4 is the most abundant CYP2J isoform in the rat lung under basal conditions.
Our study is the first to identify a decline in pulmonary CYP2J protein resulting from an infection (inflammation) caused by acute pneumonia in rats. CYP enzymes can be suppressed or induced depending on the model of inflammation, the cytokines released, and the tissues involved (26, 28, 36). Little is currently known about the regulation of CYP2J4. CYP2J4 protein is induced by pyrazole in mouse tissues, including lung (45, 53). However, little is known about the regulation of CYP2J4 in inflammation. In human and rat tissues, discordance between CYP2J mRNA and protein levels has been observed (35). Suppression of specific CYP mRNAs, caused by cytokines and other inflammatory stimuli, has been described in models of inflammation, especially for hepatic CYP isozymes (27). In addition, CYP protein turnover rates are heterogeneous and might involve ubiquitination and/or proteasome-mediated degradation, depending on the CYP isozyme involved (33). In our rat model of pneumonia, the decline in microsomal CYP2J4 protein content could be the result of a decline in transcription or translation of this enzyme or because of increased degradation of the protein caused by the proteasome degradation system.
We previously reported a decreased rate of formation of EETs in this acute pneumonia model of the rat and suggested a role for these mediators in the control of vascular tone (49). Our current findings support the hypothesis that the decrease in CYP2J4 protein contributes to this decreased level of EETs. CYP2B content is not different in lungs of pneumonia and control rats, and this isozyme is mainly present in Clara cells of the rat lungs (32). Therefore, CYP2J4 expression (and its EETs metabolites) in vascular smooth muscle cells and endothelium seems more likely to modulate pulmonary arterial tone in health and disease.
CYP enzymes play a crucial role in the modulation of vascular homeostasis (9, 14). Blood vessels demonstrate a wide range of biosynthetic capacity and diversity with regard to CYP-generated AA products. EETs are converted by vascular endothelial and smooth muscle cell epoxide hydrolases to dihydroxyeicosatrienoic acids (4, 7, 25). In comparison to prostaglandins, which require de novo synthesis, EETs and HETEs can be stored in tissue lipids, esterified to membrane phospholipids, and released in response to hormonal stimuli (5, 50). Therefore, quantitative and qualitative differences of the CYP isozymes in their distribution, induction, or suppression depend on the species, the vascular bed, and the presence of injury (12, 13, 27, 29, 31). Our study adds to the present knowledge and further stresses the importance of CYP enzymes and their AA metabolites in the control of the pulmonary circulation. As stated earlier, we did not detect significant amounts of CYP4A protein in lung microsomes from control and pneumonia rats. The lack of pulmonary expression of CYP4A is not surprising, since CYP4A is usually predominantly expressed in the rat kidney (15). In addition, in our study, we used lung microsomes prepared from pneumonia and control rat lung homogenates, whereas previous studies that identified CYP4A protein used microsomes prepared from pulmonary arteries dissected from rabbit or rat lung (54, 55). Because we previously demonstrated formation of significant amounts of 20-HETE in rat lung microsomes from control compared with pneumonia rats (49), we can speculate that other unidentified CYP enzyme(s) in microsomes of whole lung homogenates might be prominent source(s) of 20-HETE in rats.
This study demonstrates that rat lung contains a protein with immunological characteristics identical to CYP2J4 and that the content of this CYP isozyme is reduced in pneumonia. This observation supports a role for CYP2J4 in the metabolic and functional changes observed in pneumonia compared with control lungs. Therefore, we suggest that CYP2J (but not CYP2B) enzymes and their AA metabolic products contribute to the modulation of pulmonary vascular tone in rats with pneumonia.
Support for this work was provided by Canadian Institutes of Health Research Grants MT13944 (to Dr. D. G. McCormack) and FRN9722 (to Dr. J. R. Bend). A. Yaghi was supported by a fellowship from the Ontario Thoracic Society and the Ontario Graduate Scholarship in Science and Technology. Portions of this work were funded by the Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health.
We thank Dr. Laurence Kaminsky for providing the recombinant CYP2J4 used for this work. Appreciation is expressed to Viki Massey and the Microbiology Staff at the London Health Sciences Centre for providing the Pseudomonas pneumonia organisms.
Part of this work was presented at the 2001 American Thoracic Society meeting. The abstract was published in Am J Respir Crit Care Med 163 (5): A396, 2001. Also, part of this work was published as chapter 4 of the Ph.D. thesis of A. Yaghi (2002).
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.
- Copyright © 2003 the American Physiological Society