INTRODUCTION

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DESPITE EXTENSIVE investigation, the mechanisms that underlie acute hypoxic pulmonary vasoconstriction (HPV) are incompletely understood. Many factors including nitric oxide (NO) and prostanoids have been investigated as possible modulators of the acute hypoxic response of the pulmonary vasculature (28). We have recently shown that 20-hydroxyeicosatetraenoic acid (20-HETE) is an eicosanoid product of human lung tissue that acts as a potent vasodilator of isolated pressurized pulmonary arteries (PA) (1). Furthermore, cytochrome P-450 4A (CYP4A) protein is expressed in the pulmonary vasculature, a finding based upon Western blots of PA microsomes (31), conversion of arachidonic acid (AA) into 20-HETE by dispersed vascular smooth muscle cells (31), and immunohistochemistry localizing CYP4A to rabbit pulmonary capillary endothelium (20). These data raise the possibility that products of CYP4A may contribute to control of PA tone. However, the role of 20-HETE or other P-450 metabolites of AA in the pulmonary circulation is incompletely understood. We postulated that, as a vasodilator, 20-HETE might counter increases in PA tone produced by conditions such as hypoxia. To address these questions, we studied the effect of inhibition of CYP4A enzymes with two mechanistically dissimilar - and 1-hydroxylase inhibitors, 17-oxydecanoic acid (17-ODYA) and N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), on the acute hypoxic vasoconstrictive response in isolated blood-perfused rabbit lungs. Our data show that inhibition of 20-HETE formation by both drugs in this model enhances the acute hypoxia-induced increase in pulmonary perfusion pressures. 20-HETE relaxes rabbit PA rings, and inhibitors of 20-HETE shift the concentration response of PA rings to phenylephrine (PE) to the left, consistent with loss of a prorelaxing metabolite. Finally, CYP4A immunospecific protein as well as 20-HETE formation in lung microsomes from male rabbits suggest that 20-HETE is an endogenous metabolite of rabbit lungs that could modulate PA tone.

	METHODS

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Reagents. 20-HETE, 17-ODYA, DDMS, and 6-(20-propargyloxyphenyl)hexanoic acid (PPOH) were synthesized by J. R. Falck. [¹⁴C]AA was acquired from DuPont New England Nuclear, Wilmington, DE. Western blots were visualized using Renaissance Western blot chemiluminescence reagent, DuPont New England Nuclear. All chemicals were of analytical grade unless otherwise stated.

Isolated perfused lung studies. The protocol for animal use was approved by the Animal Care and Use Committee of the Medical College of Wisconsin. Perfused lungs were prepared as previously described (15). New Zealand White male and nonpregnant female rabbits (2-4 kg) were anesthetized with pentobarbital sodium (50 mg/kg). A catheter was placed into a carotid artery, and the animal was exsanguinated. After an endotracheal tube was placed, the chest was opened, and catheters were positioned in the PA and left atrium. The heart and lungs were removed and ventilated (Harvard pump) through the endotracheal tube at 50 breaths/min. End-inspiratory and end-expiratory pressures were maintained at 10 and 1 cmH₂O, respectively, by adjusting tidal volume (~20 ml/min) and were maintained by means of a water-seal pressure overflow. The lungs were ventilated with a gas mixture appropriate to the experimental protocol; FI_O2 = 0.21 for normoxia and 0.05 for hypoxia. Both gas mixtures contained 5% CO₂ with the balance being nitrogen. The collected blood was adjusted to a packed cell volume of 30-35% and added to a heated (37°C) recirculating system. The volume of blood in the reservoir was 50 ml. Blood from the recirculating system was pumped (peristaltic pump, Masterflex) into the PA at a flow rate of 100 ml/min. Left atrial pressure was maintained at 0.5-1 mmHg by adjusting the height of the venous outflow collection reservoir. PA pressures were measured and recorded continuously using CODAS software, at a rate of 10 samples/s, or a chart recorder. After baseline values of pressures and arterial blood gases were obtained, the preparation was ventilated with hypoxic gas mixtures for 3-min periods, with these exposures being repeated three to six times. Between exposures to hypoxic gases, lungs were ventilated with normoxic gases for 10-15 min, during which time return to stable baseline values was established. After three to four "control" hypoxic exposures, either 17-ODYA (final concentration 10 µM), DDMS (50 µM final concentration), or vehicle (ethanol 0.2%) was added to the preparation by injection into the PA. Two additional exposures to hypoxia were performed following injection of drug or vehicle alone, and data from the 3rd and 4th "control" or "treatment" episodes were averaged (n of 2 or 3 under each condition).

PA ring studies. PA rings were obtained and examined for isometric contractile responses according to methods previously described for study of bronchial tone (9). Briefly, male rabbits were anesthetized, the heart and lungs were removed as above, and PA rings 1-2 mm in diameter were dissected free in ice-cold buffered saline solution. Rings were mounted on tungsten wires, with one wire connected to a fixed holder and another wire connected to a force displacement transducer (model FT03, Gould Electronics) for continuously measuring isometric tension, and immersed in pH-adjusted, oxygenated physiological saline solution (PSS) at 37°C. Tension data were relayed from transducers to a signal amplifier (600 series, 8-channel amplifier, Gould Electronics). Data were acquired and analyzed using CODAS software (DataQ Instruments). Rings were loaded with 0.75 g passive tension and then equilibrated for an additional 30 min before the studies were begun. Viability of the rings was determined by measuring the contractile response to the addition of 12.5 and 25 mM KCl to the bath. We examined the concentration response of rings to incremental concentrations of PE (10¹⁰ to 10⁶ M) pretreated with vehicle, 10 µM 17-ODYA, or 1 µM PPOH, as well as the effect of 20-HETE on the tone of PA rings preconstricted with PE.

Cytochrome P-450 metabolism of AA by lung microsomes. Microsomes were prepared from homogenates by differential centrifugation in a modification of methods previously reported by us (1). Protein was quantified according to the method of Bradford (2). Microsomal proteins (1 mg/ml, 200 µl final volume) were resuspended in assay buffer (100 mM KPO₄, 1 mM EDTA, and 10 mM MgCl₂) and incubated for 60 min at 37°C with [1-¹⁴C]AA (0.125 µCi/ml; 5 µM). NADPH (1 mM) and a NADPH regenerating system containing 10 mM isocitrate and 0.1 U/ml isocitrate dehydrogenase (6) were included in each assay. Assays were performed in the presence of room air (PO₂ ~ 140 mmHg), nitrogen for hypoxic experiments (PO₂ ~ 20 mmHg measured by oxygen electrode), or a controlled blend of the two with PO₂ measured by oxygen electrode. Reactions were terminated by acidification with 1 M formic acid, and the product was extracted twice with ethyl acetate. The organic phase was back-extracted with 1 ml distilled water, evaporated under nitrogen, and reconstituted in ethanol. Reaction products were separated on a C-18 reverse phase HPLC column (Supelco, Bellefonte, PA) 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. ¹⁴C-labeled products were detected using a radiation detector (HPLC, Beckman System Gold programmable detector module no. 171). Identification of metabolites was based upon coelution with authentic standards. Authentic 20-HETE (derived from pregnant rabbit lungs incubated with [¹⁴C]AA, separated by HPLC, and verified by GC mass spectrometry, or synthesized by J. R. Falck) had a retention time of 22-23 min in our system.

Western blot identification of CYP4A protein. Microsomal suspensions were separated by electrophoresis on 10% SDS-PAGE gels and transferred to a nitrocellulose membrane. Nonspecific binding was blocked by incubating the membrane overnight in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) plus 10% nonfat milk. The membrane was then incubated for 2 h at room temperature with a polyclonal antibody (1:2,000) to rat liver CYP4A -hydroxylase enzyme that cross-reacts with 4A1, 4A2, and 4A3 isoforms (6). Last, the membrane was incubated with horseradish peroxidase-labeled secondary antibody (1:1,000) and then visualized using enhanced chemiluminescence.

Statistics. Data are presented as means ± SE. Differences between perfusion pressures during hypoxia with 17-ODYA or vehicle and during normoxia after treatment with 17-ODYA or vehicle were assessed using one-way ANOVA for repeated measures followed by a Student-Newman-Keuls test when significant differences were identified. P  0.05 using a two-tailed test was considered significant. ED₅₀ for 17-ODYA was calculated from the best fit of the data to a single exponential decay using SigmaPlot Scientific Graphing Software (Jandel).

	RESULTS

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Data pertaining to rabbits used for isolated perfused lung studies appear in Table 1. Animals were divided roughly equally between the sexes, and when examined separately, there were no significant differences in data obtained in male and female rabbits (data not shown). Therefore, results were pooled and presented together. Partial pressures of oxygen in the gases used for ventilation of isolated perfused lungs were 108 ± 0.8 mmHg in normoxia and 37 ± 0.8 mmHg in hypoxia. Mean PO₂ of circulated blood during ventilation with normoxic gas was 107 ± 2.4 mmHg and was 40 ± 1.4 mmHg during ventilation with hypoxic gases. Repeated measures of blood gases during hypoxic exposures and following return to normoxic conditions demonstrated good stability of the preparation during experiments which continued for ~2 h (see Table 1). In addition, wet-to-dry weights of lungs demonstrated no accumulation of significant extravascular lung water during the course of the experiments.

The baseline changes in perfusion pressure during hypoxic ventilation were modest (see Fig. 1, A and B), particularly during the first hypoxic period. Peak responses to hypoxia increased incrementally from the 1st through the 3rd exposures in vehicle-treated preparations. In contrast, responses in the 3rd through the 5th exposures were not different from one another (Fig. 1A). Therefore, peak responses occurring during the 3rd through 5th hypoxic episodes after vehicle were averaged to compare with responses observed after 17-ODYA or DDMS. Perfusion pressures increased from a baseline of 10 ± 0.7 to 15 ± 0.7 mmHg (n = 14) upon switching to ventilation with hypoxic gas mixtures in preparations during the control period (Fig. 1C). No changes in baseline or peak hypoxic responses (exposures 3-5) were evident after administration of vehicle alone. Administration of 17-ODYA or DDMS increased baseline (normoxic) pressures over that of baseline or vehicle control values. Furthermore, changes in perfusion pressure in response to hypoxia were significantly increased after treatment with the suicide substrate hydroxylase inhibitors of 20-HETE synthesis, 17-ODYA or DDMS (Fig. 1C).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   A: relationship between increase in pulmonary artery perfusion pressures (PAP) during ventilation with hypoxic gas and sequential exposures. Data are mean values with standard error bars, with the number of experiments indicated inside the bars. First exposures elicited a very small increase in perfusion pressures (<1 mmHg). In contrast, perfusion pressures increased by more than 5 mmHg by the 3rd exposure. Differences in perfusion pressure increases between the 3rd and 5th hypoxic exposure were not significant. B: a representative tracing of perfusion pressures in an isolated rabbit lung preparation upon switching to ventilation with hypoxic gas mixtures after treatment with vehicle (top, 3rd exposure to hypoxia) or 17-oxydecanoic acid (17-ODYA) (bottom, 4th exposure to hypoxia). Hypoxia-induced increases in perfusion pressure were amplified after treatment with 17-ODYA. C: averaged perfusion pressures in isolated perfused rabbit lungs at baseline (n = 14), after hypoxia (peak pressures from exposures 3 through 6 were averaged; n = 14), after pretreatment with vehicle (n = 4), 17-ODYA (n = 5), or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS, n = 4) alone, and finally during ventilation with hypoxic gas following treatment with vehicle (n = 4), 17-ODYA (n = 5), or DDMS (n = 4). Hypoxia alone effected a modest increase in pressures (5 ± 0.7 mmHg or 55% above baseline). #Different from baseline. Likewise, 17-ODYA or DDMS increase baseline (normoxic) perfusion pressure above that of vehicle (15 ± 5 and 22 ± 10%, respectively). *Different from vehicle. After treatment with 17-ODYA or DDMS, hypoxia caused a marked increase in perfusion pressures (105 ± 11 and 92 ± 11% above baseline for 17-ODYA and DDMS, respectively).

PA ring tension studies. We also examined the effects of 20-HETE and inhibitors of CYP4A on rabbit PA tone in vitro. 20-HETE (10⁶ M) relaxed PE-constricted rings (Fig. 2). Other concentrations of 20-HETE were not systematically studied, but decreases in tension for concentrations of 20-HETE <10⁸ M (n = 3) were not different than those elicited by vehicle alone. Concentrations of 20-HETE >10⁵ M were not tested because of confounding vehicle effects.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   20-Hydroxyeicosatetraenoic acid (20-HETE) vehicle (ethanol) in volumes twice that used to deliver 20-HETE had no effect on ring tension. Phenylephrine (PE, 107 M) constricted PA rings, and 20-HETE (106 M final concentration) relaxed preconstricted rings. *Different from tone in the presence of vehicle. **Different from vehicle control and PE preconstricted rings (n = 5).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   The tension response of PA rings is plotted as a function of PE concentration in the bath. PE is a potent constrictor of PA rings. Treatment with 10 µM 17-ODYA shifts the concentration response curve to the rings to PE to the left. In contrast, treatment with 1 µM of the epoxygenase inhibitor 6-(20-propargyloxyphenyl)hexanoic acid (PPOH) shifted the concentration response to PE to the right (n = 6). *Different from tone at the indicated concentrations of PE in the presence of vehicle.

P-450 assays. Representative chromatograms of products formed when rabbit lung microsomes were incubated with AA and vehicle, 10 µM 17-ODYA, or 50 µM DDMS are shown in Fig. 4, A and B. The major metabolite was a product with a retention time of 22-23 min (identical to that of authentic 20-HETE; see Fig. 4, inset), although peaks with elution times identical to those of epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (diHETs) as well as prostanoids (products which eluted prior to 12 min) were also seen in most samples. Conversion rates of AA into 20-HETE under control conditions (normoxia, vehicle only) were 4.8 ± 0.9 pmol · mg protein¹ · min¹ (n = 4). Synthesis of this metabolite was blocked by 17-ODYA and DDMS. The ED₅₀ for blockade of 20-HETE production by 17-ODYA was 2.9 µM (Fig. 4C). In addition, conversion of AA into 20-HETE was inhibited by hypoxia, with <5% total product formation being observed in assays performed under conditions with PO₂  20 mmHg compared with those of identical microsomes incubated with room air (n = 6; Fig. 5). In assays performed with microsomes selected for optimal epoxygenase activity, conversion of AA into products that coeluted with EETs and diHETs (as well as those comigrating with 20-HETE) was blocked by >80% in the presence of 50 µM DDMS or 10 µM 17-ODYA (data not shown). In the same microsomes, 10 µM PPOH selectively blocked EET production by >70% while having minimal effect on 20-HETE formation (<10% inhibition, data not shown). Finally, neither DDMS nor 17-ODYA changed rates of synthesis of cyclooxygenase or lipoxygenase products.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Representative chromatograms from pairs of peripheral lung microsomal proteins incubated with vehicle (A, top) or 17-ODYA (A, bottom), 10 µM final concentration, and vehicle (B, top) or DDMS (B, bottom), 50 µM final concentration. The dominant eicosanoid product has a retention time of 22-23 min. This product comigrates with authentic 20-HETE (see A, inset; labeled 20-HETE is added to an assay sample incubated with 100 µM 17-ODYA) and is blocked by inclusion of 17-ODYA in the incubation media. B: effective inhibition of 20-HETE formation by DDMS. C: 20-HETE synthesis as a function of 17-ODYA concentration in the assay solution. The line represents the best fit of a single exponential decay to the data. Half block in microsomal preparations from 4 rabbit lungs studied in at least 6 concentrations of 17-ODYA was 2.9 µM. CPM, counts per minute; DHETs, diHETES; AA, arachidonic acid.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   The relative conversion of AA into 20-HETE is plotted as a function of oxygen tension in the assay environment. Data represented with the same symbol depict values from a single microsomal preparation. Hypoxia reduced conversion of substrate to 20-HETE with an ED50 of ~80 mmHg.

Immunospecific protein identification. Rabbit lung microsomes demonstrated CYP4A immunospecific protein bands at ~49 and 53 kDa (see Fig. 6A). A fainter band of slightly larger molecular mass (~54 kDa) was seen in microsomes from two female rabbits (lanes 2 and 4, Fig. 6A). Rabbit renal microsomes, a recognized rich source of CYP4A protein, demonstrate the same banding pattern (although mostly without the largest molecular mass isoform) as lungs. These data confirm the presence of CYP4A protein in the lungs of rabbits similar to those used for the isolated perfused studies described above. Because the primary antibody was raised against rat liver CYP4A protein, we also compared immunospecific patterns of rat and rabbit lung microsomes (Fig. 6B). Rat lung microsomes demonstrated two distinct immunospecific bands, ~50 and 52 kDa, consistent with the size of 4A2 and 4A3 previously described in rat renal cortex (12).

View larger version (81K): [in this window] [in a new window]   Fig. 6.   A: immunospecific protein in lung microsomes from 4 rabbit peripheral lung microsomes (left) alongside microsomes from 2 rabbit kidneys (right) for comparison. Lung microsomes in lanes 2 and 4 were obtained from female rabbits, whereas those from lanes 1 and 3 were derived from male rabbits. Western blots from kidney microsomes were studied because the kidney is a rich source of CYP4A. Proteins (80 µg each for lung and 40 µg for renal microsomes) were separated electrophoretically on a 10% SDS-PAGE and probed with a primary polyclonal antibody raised against rat liver CYP4A1 (with cross-reactivity to 4A2 and 3). Two bands of ~49 and 53 kDa are visible in lung and renal microsomes. A faint third band of slightly larger molecular weight is apparent in Western blots from microsomes of two female rabbits studied, although gender differences in immunospecific protein expression were not systematically studied. B: microsomal proteins from the peripheral lungs of 2 male rabbits and 2 rats separated electrophoretically and probed with our polyclonal antibody to CYP4A1 (with cross-reactivity to 4A2 and 3) as in A. In rats as well as rabbits, 2 bands in each lane are easily distinguished, although each of the bands in rats migrates in a manner suggesting a slightly different molecular weight than the proteins identified by the same primary antibody in rabbits. The two bands in rat lung are consistent with the size of CYP4A2 (50.5 kDa) and 4A3 (52 kDa) known to be expressed in rat kidney and liver (12).

	DISCUSSION

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We previously reported that 20-HETE is produced by microsomes prepared from human lungs and that it is a potent dilator of isolated human PAs (1). These data suggest that 20-HETE could function to maintain low pulmonary vascular resistances at rest or to counter increases in PA pressures, such as might be experienced with exercise or hypoxia. In the present study, we therefore examined the contribution of 20-HETE to HPV in isolated perfused rabbit lungs. We found that HPV is amplified severalfold in isolated perfused lungs that are treated with either of two structurally and mechanistically different inhibitors of 20-HETE formation, 17-ODYA or DDMS. These data suggest that P-450 metabolites of AA oppose hypoxic vasoconstriction.

However, neither 17-ODYA nor DDMS has a sufficiently narrow spectrum that we can attribute enhanced hypoxic vasoconstriction after treatment with these agents to blocked formation of a specific metabolite based upon data from the isolated perfused lung model alone. Both 17-ODYA and DDMS can inhibit the synthesis of epoxygenase products (EETs) at concentrations needed to inhibit 20-HETE formation in vivo (e.g., Refs. 16, 27, and our data). EETs (as well as 20-HETE) are endogenous products of rabbit lung (present study and Ref. 33). Thus blunted formation of either 20-HETE or EETs might account for 17-ODYA- or DDMS-augmented hypoxic vasoconstriction in isolated perfused lungs. For this reason, we performed additional experiments to address the potential contribution of 20-HETE vs. EETs to PA tone. We found that 20-HETE relaxes PE-preconstricted rabbit PA rings in a concentration-dependent manner. Moreover, the concentration response of rings to PE is shifted to the left by 20-HETE inhibition, consistent with the interpretation that 17-ODYA blocks synthesis of a factor which decreases PA tension. We also found that PPOH, a selective inhibitor of epoxygenases (EETs; Ref. 16 and our data) shifts the PE concentration response of PA rings to the right, suggesting that EETs have the opposite effect of 20-HETE and increase the state of activation of rabbit PA rings. These observations are in keeping with our previous observations that EETs constrict isolated pressurized rabbit PA in a concentration-dependent manner (32). Together with the data from isolated perfused lungs, our findings in PA rings suggest that 20-HETE dilates PA and that blockade of endogenous synthesis of this vasorelaxant metabolite by 17-ODYA or DDMS contributes to the augmented constriction of isolated perfused lungs upon acute exposure to hypoxic ventilation.

There is good evidence that hypoxia inhibits the opening of potassium-selective channels in PA smooth muscle cells and that hypoxia is associated with increases in intracellular calcium due to influx through voltage-activated calcium channels and release of calcium from the sarcoplasmic reticulum (e.g., Ref. 28). However, the mechanisms by which hypoxia effects these changes are uncertain. NO (endothelium-derived relaxing factor, EDRF) and prostacyclin also modulate HPV (18, 21, 23, 26). NOS inhibition in healthy human volunteers augments hypoxia-induced increases in pulmonary vascular resistance (2). NO synthase inhibitors and indomethacin increase the acute vasoconstrictive response to hypoxia in rabbit and dog lungs, similar to the effect that we observed with 17-ODYA treatment (23, 24). We interpret these data to mean that NO, prostacyclin, and 20-HETE (present study) cannot be the mediators of the abrupt rise in pulmonary vascular resistance evoked by hypoxia, because inhibited formation of these metabolites amplifies rather than blunts the response. Several investigators have reported interactions between NO and P-450 metabolites, whereby binding of NO to the heme moiety of cytochromes P-450 and blockade of 20-HETE formation may account for some hypoxia-induced relaxation of systemic arteries (10, 15). Chang et al. (4) reported blunted initial rises in PA pressures of hypoxic ventilated rat lungs pretreated with the P-450 inhibitor 1-aminobenztriazole (1-ABT). Similarly, Weissmann and colleagues (29) found attenuated hypoxic constriction in buffer-perfused rabbit lungs pretreated with 1-ABT, methoxsalen, or nordihydroguaiaretic acid (a lipoxygenase/cyclooxygenase inhibitor). Other investigations have demonstrated that 1-ABT and its derivatives are nonspecific inhibitors, blocking CYP1A1, 2B, and others (22). Our data using more specific CYP inhibitors in blood-perfused rabbit lungs, complemented by microsomal assays to evaluate product formation with inhibitors and hypoxia, and additional experiments with PA rings suggest that CYP4A metabolites can modify the pulmonary vasoconstrictive response to hypoxia.

CYP4A products and proteins were first identified in rabbit lungs nearly 20 years ago (19, 30). Expression of CYP4A isoforms and production of 20-HETE are induced by pregnancy (>100-fold) or progesterone therapy (13, 14, 20). However, the conversion of AA to 20-HETE can be easily detected in lung microsomes prepared from male rabbits (31, 33). Rabbit kidneys are reported to express at least two CYP4A isoforms (11, 17). Originally referred to as P-450a and P-450b, these enzymes have monomeric molecular masses of 53 and 49 kDa, respectively. In the present study, we utilized a primary antibody raised against rat liver CYP4A1, with cross-reactivity to 4A2 and 4A3, and found expression of the CYP4A protein in both kidney and lung microsomes. In female rabbits, a band of slightly greater molecular mass was evident, consistent with expression of another CYP4A4-like protein or alternative sliced variant of one of the other CYP4A isoforms, although our investigations were not designed to examine gender differences in isoform expression. Our data provide evidence that at least two or more CYP4A isoforms are expressed in male and female rabbit lungs.

The present study also demonstrates that 20-HETE synthesis in lung microsomes is decreased to nearly undetectable levels in hypoxic environments, qualitatively similar to data previously reported in rat renal arterioles (7). CYP4A enzymes are regulated in a manner opposite to that of other pulmonary vasodilator products by hypoxia; i.e., endogenous production of prostacyclin and NO are upregulated by subacute or chronic hypoxia (21). COX-2 (and not COX-1) transcripts are increased by 3 h of hypoxic exposure in isolated perfused rat lungs (5), as is the release of NO from the pulmonary circulation in rats exposed to chronic hypoxia (8). Vasodilators support perfusion to hypoventilated regions of the lung, thus worsening systemic hypoxemia (e.g., Ref. 24). Therefore, we speculate that in addition to blunting or countering acute hypoxia-induced increases in pulmonary vascular tone, inhibited synthesis of 20-HETE under conditions of chronic hypoxia might afford a mechanism to divert blood flow away from localized areas of lung exposed to persistent hypoventilation, thus better matching ventilation and perfusion.

In conclusion, our data demonstrate that rabbit lung microsomes metabolize AA into 20-HETE in a process that can be inhibited by 17-ODYA, DDMS, and hypoxia. Furthermore, they demonstrate immunospecific CYP4A protein. Inhibition of CYP4A with two chemically and mechanistically different inhibitors amplifies the vasoconstrictive response of isolated perfused rabbit lungs to hypoxia. In addition, 20-HETE relaxes rabbit PA rings, and inhibition of 20-HETE formation shifts the concentration response to PE to the left. All of these observations are consistent with prodilatory actions of 20-HETE on rabbit PA. These data confirm and extend our previous finding that 20-HETE is a potent vasodilator in isolated human PAs (1) and suggest that endogenous production of this metabolite may modify PA tone, particularly under conditions of hypoxia.