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We previously reported that the levels of epoxyeicosatrienoic acids (EETs) and 20- ... (CYP) metabolites of arachidonic acid (AA) in contractile responses of both ...
Articles in PresS. Am J Physiol Lung Cell Mol Physiol (July 25, 2003). 10.1152/ajplung.00039.2003 Revised manuscript # LCMP-00039-2003 R1

FINAL ACCEPTED VERSION Pulmonary cytochrome P450 2J4 (CYP2J4) is reduced in a rat model of acute Pseudomonas pneumonia Asma Yaghi1, 2, J. Alyce Bradbury3, Darryl C. Zeldin3, Sanjay Mehta1, 2 John R. Bend2 and David G. McCormack1, 2

Affiliation: 1

AC Burton Vascular Biology Laboratory, Lawson Health Research Institute, Respirology, London Health Sciences Centre, Victoria Campus, 375 South Street, London, Ontario, Canada, N6A 4G5.

2

Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada, N6A 5C1.

3

National Institute of Environmental Health Sciences, NIH, Research Triangle Park, N.C. 27709, USA

Running head: CYP2J4 is reduced in a Rat Model of Acute Pneumonia Correspondence: Dr. David G. McCormack London Health Sciences Centre, Victoria Campus 375 South Street London, Ontario Canada N6A 4G5 Phone: FAX: E-MAIL:

(519)-667-6767 (519)-667-6687 [email protected]

Keywords: CYP450; inflammation; western blotting; CYP2J4 and CYP4A protein content; peptide antibodies.

Copyright (c) 2003 by the American Physiological Society.

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Abstract We previously reported that the levels of epoxyeicosatrienoic acids (EETs) and 20hydroxyeicosatetraenoic acid (20-HETE) are depressed in microsomes prepared from lungs of rats with acute Pseudomonas pneumonia. We also showed a potential role for cytochrome P450 (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 into the lungs of rats. Forty-four hours later, 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), antiCYP2J2pep4, or anti-CYP2J2pep3 (both of which react with all known CYP2J isozymes). Western blotting revealed a prominent 55kDa band with anti-CYP2J2pep3, anti-CYP2J2pep4 and anti-CYP2J6pep1 (but not anti-CYP2J9pep2) that was reduced in pneumonia compared to control lung microsomes. The CYP2B bands (51-52kDa) 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 immunologic characteristics similar to CYP2J4 and this CYP is reduced following 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.

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Introduction There is considerable evidence in the literature implicating the cytochrome P450 (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 following 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 to 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 due to 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

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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 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 immunologic characteristics similar to CYP2J4. Further 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 to 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

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for fluid administration. Animals in the pneumonia group were injected intratracheally with 0.15 ml saline containing 3Χ108 colony forming units/ml through a tracheostomy. Within 36 hours, 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 (1U/ml) at 2 ml per hour. Fentanyl, 1.0 µg/ml, was added to the venous infusion for analgesia. Forty-four hours following surgery, rats were anesthetized with pentobarbital (30 mg/kg, intravenously), and the thorax was opened. The heart and lungs were removed en bloc and perfused through the pulmonary artery with Krebs' bicarbonate solution (mM: NaCl, 118; KCl, 4.7; CaCl2, 2.5; MgSO4.7H2O, 1.2; KH2PO4, 1.2; dextrose, 11.1; NaHCO3, 22.1; 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 naïve lungs were also prepared. Naïve 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. (1951) using bovine serum albumin as the standard.

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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 (antiCYP2J2pep4) were raised in rabbits and cross-react with all known CYP2J subfamily P450s. None of these antibodies are known to cross-react with non-CYP2J subfamily P450s. Preimmune serum, collected from the rabbits prior to 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 onto polyvinylidene difluoride (PVDF) membranes (Amersham Canada Ltd., Oakville, ON). Membranes were immunoblotted using anti-CYP2B1/2B2 (dilution: 1/100; Cedarlane Laboratories ltd., Hornby, ON, Canada), antiCYP2J9pep2, anti-CYP2J6pep1, anti-CYP2J2pep4, or anti-CYP2J2pep3. All the CYP2J antibodies were used at a dilution of 1/1000. Bound antibodies were detected using goat anti-

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rabbit IgG conjugated with horseradish peroxidase (dilution: 1/2000; Promega Corporation, Madison WI, USA) and the enhanced chemiluminescence western blotting detection system (ECL Plus, Amersham Canada Ltd., Oakville, ON). Relative molecular weights of proteins were determined by running a prestained protein marker (Promega Corporation, Madison, WI, USA) 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 (BioRad Laboratories, Inc., Hercules, CA, USA) was used for measuring optical densities. Band densities were expressed as adjustable volumes (O.D.Xmm2 = optical density multiplied by area in mm squared). 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 two hours. Membranes were then washed in TBS-T (washing buffer: Tris-buffered saline with 0.1% Tween 20), dipped in polyvinyl alcohol (1 mg/500 ml blocking buffer TBS, stored at 4 °C), and blocked for 2 hours in Blocker Blotto in TBS (Pierce, Rockford, IL, USA). Western blotting was continued beginning with primary antibody. Stripping buffer composition (100 ml) was as follows: 6.25 ml 1M Tris (pH 6.7), 20 m1 of 10% SDS, 0.69 m1 of 2-mercaptoethanol (14.4M), and 73.06 ml distilled water. CYP4A A similar method was followed for western blotting as above. PVDF membranes were immunoblotted with anti-rat CYP4A polyclonal antibody (Chemicon International Inc., Temecula, CA, USA) at 1:1500 dilution. Rat kidney microsomes were used as a positive control for CYP4A isozymes.

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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 Ltd. (Oakville, ON). Blotto in TBS (Pierce, Rockford, IL, USA) was used for blocking the PVDF membranes. Data analysis Densitometry results are expressed as mean ± standard error of the mean of n values, where n = number of rats. Results were compared using ANOVA followed by Bonferroni’s post-tests. A value of p