Cytosolic phospholipase A₂ in hypoxic pulmonary vasoconstriction

Cytosolic phospholipase A₂ (cPLA₂) releases arachidonic acid (AA) from phospholipids in cell membranes. To assess the role of cPLA₂ in hypoxic pulmonary vasoconstriction (HPV), we measured the increase in left lung pulmonary vascular resistance (LPVR) before and during hypoxia produced by left main stem bronchus occlusion (LMBO) in mice with and without a targeted deletion of the PLA2g4a gene that encodes cPLA_2α. LMBO increased LPVR in cPLA_2α^+/+ mice but not in cPLA_2α^–/– mice. cPLA_2α^+/+ mice were better able to maintain systemic oxygenation during LMBO than were cPLA_2α^–/– mice. Administration of a cPLA₂ inhibitor, arachidonyl trifluoromethyl ketone, blocked the LMBO-induced increase in LPVR in wild-type mice, while exogenous AA restored HPV in cPLA_2α^–/– mice. Intravenous angiotensin II infusion increased PVR similarly in cPLA_2α^+/+ and cPLA_2α^–/– mice. Inhibitors of cyclooxygenase or nitric oxide synthase restored HPV in cPLA_2α^–/– mice. Breathing 10% oxygen for 3 weeks produced less right ventricular hypertrophy in cPLA_2α^–/– than in cPLA_2α^+/+ mice, but restored HPV in cPLA_2α^–/– mice despite the continued absence of cPLA₂ activity. These results indicate that cPLA₂ contributes to the murine pulmonary vasoconstrictor response to hypoxia. Augmenting pulmonary vascular tone restores HPV in the absence of cPLA₂ activity.

Hypoxic pulmonary vasoconstriction (HPV) is an intrinsic property of pulmonary vascular smooth muscle that produces vasoconstriction in poorly ventilated lung regions (1). This important mechanism acts to enhance the matching of ventilation with perfusion and maintain arterial oxygenation in lung disease. Despite intensive investigation, a comprehensive understanding of the cellular mechanisms that underlie HPV remains elusive.

In the lung, the metabolism of arachidonic acid (AA) by cyclooxygenases (COX), lipoxygenases (LOs), and cytochrome P450 (CYP450) enzymes generates a wide variety of eicosanoids, including prostaglandins, thromboxanes, leukotrienes, epoxyeicosatrienoic acids (EETs), and hydroxyeicosatetraenoic acids (HETEs), that importantly regulate vascular tone (for recent reviews see refs. 2, 3). Potential roles for eicosanoids in HPV have been studied extensively with conflicting results reported. For example, administration of exogenous AA has been shown to either attenuate (4) or enhance HPV (5). Moreover, studies of HPV using inhibitors of COX, LOs, and CYP450 enzymes are limited by the incomplete specificity of the inhibitors, as well as the metabolic diversion of AA to alternate pathways.

Unesterified AA is present at very low levels within cells, and its rate of formation generally controls the biosynthesis of bioactive eicosanoids (2). Release of AA from the sn-2 position of membrane phospholipids is mediated by phospholipase A₂ (PLA₂) enzymes. PLA₂ enzymes can be classified into three main types based on biological properties, including secretory PLA₂ (sPLA₂), cytosolic PLA₂ (cPLA₂), and intracellular Ca²⁺-independent PLA₂ (iPLA₂). Here, we studied group IVA cPLA₂, because AA generated by this PLA₂ is tightly linked to the COX and LO pathways (6) and this PLA₂ is known to be expressed in murine lung (7).

To assess the contribution of cPLA₂ to HPV, we compared the increase in left lung pulmonary vascular resistance induced by selective left lung hypoxia in wild-type mice and cPLA₂-deficient mice. Here, we report that an absence of cPLA₂ activity prevents HPV in mice.

Unilateral alveolar hypoxia and the contribution of cPLA₂. To assess the contribution of cPLA₂ to HPV, we examined changes of LPVR in response to LMBO in cPLA_2α+/+ and cPLA_2α–/– mice. Before LMBO, hemodynamic parameters did not differ between the two genotypes (Table 1). LPVR was similar in cPLA_2α+/+ mice (107 ± 13 mmHg/ml/min/g) and cPLA_2α–/– mice (97 ± 6 mmHg/ml/min/g). In cPLA_2α+/+ mice, LMBO decreased QLPA without changing PAP, more than doubling LPVR (216 ± 29 mmHg/ml/min/g; P < 0.005; Table 1 and Figure 1a). In contrast, LMBO did not change LPVR in cPLA_2α–/– mice (100 ± 10 mmHg/ml/min/g; Figure 1b), consistent with impaired HPV. In a subset of animals of each genotype (n = 3–5), QLTAF was measured as an estimate of cardiac output: LMBO did not alter QLTAF or TPVR in either genotype, demonstrating that the LMBO-induced changes in LPVR cannot be attributed to alterations in cardiac output.

Figure 1

The left lung pulmonary blood flow-pressure relationship before (baseline) and after 5 minutes of LMBO in (a) cPLA_2α^+/+ mice (n = 11) and (b) cPLA_2α^–/– mice (n = 10). The increase of the slope of the left lung pulmonary blood flow-pressure relationship after LMBO in cPLA_2α^+/+ mice represents the LMBO-induced increased in LPVR (P < 0.01 vs. baseline). In contrast, in cPLA_2α^–/– mice, LMBO did not increase LPVR (b).

Table 1

Hemodynamic measurements

To estimate the impact of impaired HPV on arterial oxygenation, we performed arterial blood gas analysis at the end of HPV measurements during LMBO while the right lung was ventilated at F_IO₂ = 1. Arterial partial pressure of oxygen (PaO₂) was significantly higher in cPLA_2α+/+ mice than in cPLA_2α–/– mice (298 ± 46 and 159 ± 23 mmHg, respectively; P < 0.05; Table 2). There was no difference in blood pH, arterial partial pressure of carbon dioxide (PaCO₂), or HCO₃^– (Table 2). These observations are consistent with increased intrapulmonary shunting in cPLA_2α–/– mice. Next, we tested whether or not pharmacological inhibition of cPLA₂ impaired HPV in wild-type mice. The LMBO-induced increase in LPVR was markedly attenuated when mice were studied 30 minutes after treatment with the cPLA₂ inhibitor, ATK, as compared with mice treated with vehicle alone (Figure 2). These results confirm that decreased cPLA₂ activity is associated with impaired HPV in mice.

Figure 2

LMBO-induced increase in LPVR is shown for cPLA_2α^+/+ (n = 11) and cPLA_2α^–/– (n = 10) mice at baseline and for wild-type C57BL/6 mice treated with vehicle (Veh; n = 5) or with ATK (n = 5). LMBO-induced increase in LPVR is also shown for mice during AA administration (n = 3 for cPLA_2α^+/+ and n = 6 for cPLA_2α^–/–), after administration of indomethacin (Indo; n = 4 for cPLA_2α^+/+ and n = 5 for cPLA_2α^–/–), after administration of L-NAME (n = 3 for cPLA_2α^+/+ and n = 5 for cPLA_2α^–/–), and after breathing at F_IO₂ 0.10 for 3 weeks (3wk Hypoxia; n = 6 for cPLA_2α^+/+ and n = 10 for cPLA_2α^–/–). *P < 0.05 vs. cPLA_2α^–/– mice at baseline. **P < 0.05 vs. cPLA_2α^+/+ mice at baseline.

Table 2

Arterial blood gas analysis

Since cPLA₂ is believed to be a major enzymatic source of AA for eicosanoid biosynthesis (3, 20) in most mammalian cells, we examined whether administration of AA restores HPV in cPLA_2α–/– mice. After continuous intravenous infusion of AA at 1 μg/kg/min for 60 minutes, LMBO increased LPVR in cPLA_2α–/– mice (Figure 2). In contrast, when studied in cPLA_2α+/+ mice, the magnitude of the LMBO-induced increase of LPVR was not altered by exogenous AA. These results suggest that impaired HPV induced by cPLA₂ deficiency was attributable to decreased availability of AA in the hypoxic lung of cPLA_2α–/– mice.

To examine the specificity of the effects of cPLA₂ deficiency on HPV, we examined whether cPLA₂ deficiency attenuates the normoxic pulmonary vasoconstrictor response to intravenous infusion of 0.05, 0.5, and 5 μg/kg/min of angiotensin II. TPVR increased from 74 ± 12 mmHg/ml/min/g at baseline to 177 ± 18 mmHg/ml/min/g at 5 μg/kg/min angiotensin II in cPLA_2α+/+ mice (P < 0.01) and from 69 ± 13 to 217 ± 39 mmHg/ml/min/g in cPLA_2α–/– mice (P < 0.001). TSVR increased from 468 ± 66 mmHg/ml/min/g at baseline to 956 ± 216 mmHg/ml/min/g at 5 μg/kg/min angiotensin II in cPLA_2α+/+ mice (P < 0.01) and from 435 ± 47 to 899 ± 144 mmHg/ml/min/g in cPLA_2α–/– mice (P < 0.001). At each angiotensin II infusion dose, there was no difference in either the TPVR or TSVR between cPLA_2α+/+ and cPLA_2α–/– mice. Similarly, the pulmonary vasoconstrictor response to angiotensin II did not differ between ATK-treated and vehicle-treated wild-type mice (data not shown). Taken together, our results demonstrate that cPLA₂ deficiency attenuated pulmonary vasoconstriction to hypoxia, without causing nonspecific dysfunction of the vasomotor contractile apparatus.

Pulmonary eicosanoid profiles. To identify the major eicosanoids generated in the lungs of cPLA_2α–/– and cPLA_2α+/+ mice, we carried out profiling of eicosanoids using both ELISA and LC/MS/MS analyses. In lungs obtained from both cPLA_2α+/+ and cPLA_2α–/– mice, we identified PGE₂, TXB₂, 5S-HETE, 12S-HETE, and 15S-HETE by matching their respective mass spectra and retention times with those of authentic synthetic standards (Figure 3, a and b). Leukotriene B₄ and EETs, as well as their respective omega-oxidation products, were not present in appreciable amounts in snap-frozen lungs from either genotype (Figure 3c) (2, 21). Moreover, 20-HETE was not detected in lungs of either genotype (22).

Figure 3

LC/MS analyses of eicosanoids from cPLA_2α^–/– mouse lungs. Results show representative LC/MS/MS analyses of lung extracts obtained from at least four separate experiments. LC retention times (RT), parent ions, and daughter ions for products extracted from murine lungs were diagnostic and matched those of authentic standards (see Methods for details). (a) LC/MS/MS spectrum (RT 4 minutes) of PGE₂. Inset: Chromatogram of ion mass/charge (m/z) 333. (b) LC/MS/MS spectrum (RT 7 minutes) of TXB₂. Inset: Chromatogram of ion m/z 369. (c) LC/MS/MS spectrum (RT 12.7 min) of LTB₄-d₄ used as internal standard. Inset: Chromatogram of ion m/z 339 showing the internal standard extracted from lung and the apparent absence of m/z 335 for endogenous LTB₄. D, deuterium.

To examine the possibility that decreased levels of vasoconstrictor eicosanoids contribute to the impaired HPV in cPLA_2α–/– mice, we quantitated vasoactive eicosanoids PGE₂, PGF_2α, and TXB₂ by ELISA in cPLA_2α+/+ and cPLA_2α–/– mice after LMBO. PGE₂ levels were higher in left than in right lungs in both genotypes (P < 0.05 for both; Figure 4a). PGF_2α and thromboxane levels were greater in left than in right lungs in cPLA_2α+/+ mice (P < 0.05 for both; Figure 4, b and c) but not in cPLA_2α–/– mice. These observations suggest that COX products are increased in hypoxic left lungs of cPLA_2α+/+ mice. However, statistically significant differences were not found in the amounts of PGE₂, PGF_2α, or TXB₂ when comparing corresponding lungs of cPLA_2α+/+ and cPLA_2α–/– mice.

Figure 4

PGE₂ (a), PGF_2α (b), and TXB₂ (c) levels in right and left lung homogenates obtained from cPLA_2α^+/+ and cPLA_2α^–/– mice after LMBOand measured by ELISA. Filled bars indicate cPLA_2α^+/+ (n = 5) and open bars indicate cPLA_2α^–/– mice (n = 5). PGE₂ levels were higher in left than in right lungs in both genotypes (P < 0.05). PGF_2α and TXB₂ levels were higher in left than in right lungs in cPLA_2α^+/+ mice (P < 0.05), but not in cPLA_2α^–/– mice. There was no difference in levels of PGE₂, PGF_2α, and TXB₂ in either lung between the two genotypes.

Both indomethacin and l-NAME restored HPV in cPLA_2α–/– mice. To examine the possibility that HPV is impaired in cPLA_2α–/– mice due to an altered vasoconstrictor/vasodilator balance, we studied the effects on HPV of inhibition of endothelium-dependent vasodilators, PGI₂, and nitric oxide (NO) by indomethacin and L-NAME, respectively. It has been reported that indomethacin and L-NAME enhance HPV at doses that do not alter the baseline pulmonary vascular resistance (16). Thirty minutes after the administration of indomethacin (5 mg/kg intravenously), the heart rate was decreased in both cPLA_2α+/+ and cPLA_2α–/– mice as compared with untreated mice (591 ± 20 vs. 389 ± 23 and 567 ± 24 vs. 358 ± 23 beats per minute, respectively; P < 0.001 for each), but other hemodynamic parameters were unaffected (Table 1). The LMBO-induced increase in LPVR did not differ between indomethacin-treated and untreated cPLA_2α+/+ mice (ΔLPVR: 102% ± 42% vs. 103% ± 17%, respectively). However, in cPLA_2α–/– mice, indomethacin treatment restored the ability of LMBO to increase the LPVR (103% ± 12%, P < 0.01, vs. untreated cPLA_2α–/– mice) to the level measured in cPLA_2α+/+ mice (Figure 2).

In concordance with results from earlier studies (16, 17), 30 minutes after administration of L-NAME, baseline hemodynamic parameters, including PAP, QLPA, and SAP, were unchanged in both genotypes (Table 1). In cPLA_2α+/+ mice, L-NAME administration augmented the LMBO-induced increase in LPVR. In cPLA_2α–/– mice, L-NAME administration restored the ability of LMBO to increase LPVR (Figure 2). Taken together, these results suggest that the pulmonary vasculature of cPLA_2α–/– mice is capable of vasoconstriction in response to hypoxia when synthesis of an endothelium-dependent vasodilator is inhibited.

Chronic hypoxia in cPLA_2α–/– mice. To learn if the attenuated vasoconstrictor response to acute hypoxia in cPLA_2α–/– mice protects mice against the pulmonary vascular remodeling associated with prolonged exposure to hypoxia, we studied cPLA_2α–/– and cPLA_2α+/+ mice that breathed at either F_IO₂ 0.10 or 0.21 for 3 weeks. W_RV/W_LV+S was not different in cPLA_2α+/+ mice and cPLA_2α–/– mice breathing air (25.2% ± 1.4% and 24.2% ± 1.5%, respectively). Although prolonged hypoxia increased W_RV/W_LV+S in both genotypes, RV hypertrophy was less prominent in cPLA_2α–/– mice (30.3% ± 1.4 %) than in cPLA_2α+/+ mice (35.5% ± 1.4%; P < 0.05). The cPLA_2α+/+ mice and cPLA_2α–/– mice had similar hemoglobin levels under normoxic conditions (12.9 ± 0.5 and 12.9 ± 0.3 g/dl, respectively). Although chronic hypoxia increased the circulating hemoglobin concentration in both genotypes, cPLA_2α–/– mice developed less polycythemia than cPLA_2α+/+ mice (16.5 ± 1.6 vs. 19.0 ± 0.3 g/dl; P < 0.05). Quantitative analysis revealed that the WT of pulmonary arterial vessels with external diameter between 14 and 100 μm was similar in cPLA_2α–/– mice and cPLA_2α+/+ mice breathing at F_IO₂ 0.21 (8% ± 2% vs. 8% ± 2%). Three weeks of hypoxia increased the WT of these pulmonary vessels similarly in both groups of mice (14% ± 2% vs. 14% ± 1%). Qualitative differences in the components of the vessel walls of the two genotypes were not evident after 3 weeks of hypoxia.

Baseline PAP, QLPA, and LPVR were measured at thoracotomy in mice during ventilation at F_IO₂ 1. PAP was greater in both cPLA_2α+/+ and cPLA_2α–/– mice that breathed at F_IO₂ 0.10 for 3 weeks than in mice that breathed at F_IO₂ 0.21 (Table 1). Breathing at F_IO₂ 0.10 for 3 weeks did not increase the baseline LPVR significantly in mice of either genotype. Prolonged hypoxia did not alter the ability of LMBO to increase LPVR in cPLA_2α+/+ mice. In contrast, after 3 weeks of hypoxia, cPLA_2α–/– mice showed a robust increase of LPVR in response to LMBO, demonstrating restored HPV in cPLA_2α–/– mice (Figure 2). Of note, after breathing at F_IO₂ 0.10 for 3 weeks, cPLA_2α–/– mice had lower LPVR at baseline and during LMBO compared with cPLA_2α+/+ mice, apparently due to increased QLPA in cPLA_2α–/– mice (Table 1).

Lung homogenates obtained from normoxic cPLA_2α+/+ mice de-esterified AA from PC, whereas lung homogenates from normoxic cPLA_2α–/– mice metabolized PC only minimally (P < 0.05, Figure 5a). In contrast, lung homogenates from the normoxic cPLA_2α–/– mouse contained PLA₂ activity directed against PE in assay conditions favoring the activity of sPLA₂. PE-metabolizing PLA₂ activity was greater in cPLA_2α+/+ mice (P < 0.05 vs. cPLA_2α–/– mice; Figure 5b) most likely because cPLA₂ also acts on PE. Prolonged hypoxic exposure did not affect the activity of pulmonary cPLA₂ or sPLA₂ in either cPLA_2α+/+ mice or cPLA_2α–/– mice (Figure 5, a and b). Lung homogenates obtained from normoxic cPLA_2α+/+ mice contained iPLA₂ activity, and it was not affected by breathing at F_IO₂ 0.10 for 3 weeks (Figure 5c). The iPLA₂ activity in lung homogenates obtained from normoxic cPLA_2α–/– mice was modestly greater than that in lung homogenates from cPLA_2α+/+ mice (P = 0.055), and it was decreased by breathing at F_IO₂ 0.10 for 3 weeks (P < 0.05 vs. normoxic cPLA_2α–/– mice; Figure 5c). Taken together, chronic hypoxia restored HPV in cPLA_2α–/– mice without augmenting pulmonary PLA₂ activities.

Figure 5

Calcium-dependent PLA₂ activity using PC substrate (a) and PE substrate (b) and iPLA₂ activity (c) in lung homogenates from the cPLA_2α^+/+ and cPLA_2α^–/– mice at baseline and after 3 weeks of breathing at F_IO₂ 0.10. Note that lung homogenates from the cPLA_2α^+/+ have PC substrate-metabolizing activity (in assay conditions favoring the activity of cPLA₂), while homogenates from the cPLA_2α^–/– mice have essentially no phospholipase activity for the PC substrate at baseline or after 3 weeks of hypoxia. In contrast, the cPLA_2α^–/– mouse lung homogenate contained PE-metabolizing PLA₂ activity (in assay conditions favoring the activity of sPLA₂). Three weeks of breathing at F_IO₂ 0.10 did not alter cPLA₂ or sPLA₂ activities in either the cPLA_2α^+/+ or cPLA_2α^–/– mouse lungs. The iPLA₂ activity was modestly greater in cPLA_2α^–/– than in cPLA_2α^+/+ mice at baseline (P = 0.055) and was decreased by breathing at F_IO₂ 0.10 for 3 weeks in cPLA_2α^–/– mice (P < 0.05 vs. normoxic cPLA_2α^–/– mice).

Effects of inhibition of iPLA₂ on HPV. Since pulmonary iPLA₂ activity tended to be greater in normoxic cPLA_2α–/– mice than in cPLA_2α+/+ mice, and iPLA₂ activity in cPLA_2α–/– mice was decreased by prolonged breathing at F_IO₂ 0.1, we considered the possibility that compensatory upregulation of iPLA₂ in cPLA_2α–/– mice impaired HPV at baseline and reduction of iPLA₂ by chronic hypoxia restored HPV in cPLA_2α–/– mice. To test this hypothesis, we examined effects of BEL, a selective iPLA₂ inhibitor, on HPV. Administration of BEL or vehicle did not affect baseline hemodynamics (data not shown). Administration of BEL did not affect HPV in cPLA_2α+/+ mice (ΔLPVR = 117% ± 20% and 102% ± 18% in BEL-treated and vehicle-treated cPLA_2α+/+ mice, respectively) or in cPLA_2α–/– mice (ΔLPVR = 21% ± 10% and 28% ± 12% in BEL-treated and vehicle-treated cPLA_2α–/– mice, respectively; both P < 0.05 vs. cPLA_2α+/+ mice with the same treatment). These results suggest that it is unlikely that a compensatory upregulation of iPLA₂ is responsible for impaired HPV in cPLA_2α–/– mice.

The present study demonstrates that cPLA₂ deficiency impairs HPV in mice resulting in a reduced ability to preserve systemic oxygenation after LMBO. Restoration of HPV in cPLA_2α–/– mice with exogenous AA suggests that AA availability itself and/or one or more downstream bioactive eicosanoids contribute to HPV in wild-type mice. Although we found that cPLA_2α–/– mice are capable of producing eicosanoids (Figure 3) and could not demonstrate quantitative differences in the levels of AA-derived eicosanoids in lungs taken from cPLA_2α–/– and cPLA_2α+/+ mice (Figure 4), it is possible that vasoactive eicosanoids present at low levels and/or generated from a minority of cells within the lungs of cPLA_2α+/+ mice (and hence not detected in whole-lung extracts using LC/MS/MS or ELISA) may be absent from those cells in cPLA_2α–/– mice, thereby impairing HPV.

Alternatively, deficiency of AA production in cPLA_2α–/– mice may modulate HPV. For example, a NADPH oxidase has been implicated in the sensing of pulmonary hypoxia (23, 24). Recently, Dana et al. used a cPLA₂-deficient cell line and showed that cPLA₂ and released AA are required for NADPH oxidase activation (25). It is possible that administration of AA restored HPV in cPLA_2α–/– mice by activating NADPH oxidase-related events.

Impaired HPV in the cPLA_2α–/– mice may be due to an alteration in the pulmonary vasoconstrictor/vasodilator balance, and administration of exogenous AA may have restored “balance.” This hypothesis is supported by our observations that both indomethacin and L-NAME restored HPV in cPLA_2α–/– mice using doses that do not alter baseline values of PAP and QLPA. Given the increased levels of COX products we detected in the left lungs after LMBO (Figure 4), the restoration of HPV by indomethacin and L-NAME is likely to be attributable to inhibition of endothelium-dependent production of vasodilator prostaglandins (i.e., PGE₂ and PGI₂) and NO, respectively, thereby shifting the “balance” in favor of vasoconstriction (16). These observations suggest that the pulmonary vasculature of cPLA_2α–/– mice remains capable of constricting in response to hypoxia after major vasodilator metabolic pathways are blocked.

Because agents that attenuate HPV can prevent pulmonary hypertension and vascular remodeling in chronically hypoxic animals (26–28), we studied the effects of chronic hypoxia on cPLA_2α+/+ and cPLA_2α–/– mice. Breathing at F_IO₂ 0.10 for 3 weeks increased PAP similarly in both mouse genotypes. In addition, pulmonary vascular remodeling was evident in both cPLA_2α+/+ and cPLA_2α–/– mice, and there was no difference in pulmonary vascular wall thickness between these genotypes. The observation that cPLA₂ deficiency partially protects against the development of RV hypertrophy in chronically hypoxic mice is potentially attributable to multiple factors. The initial absence of HPV, as well as the more modest increase in PVR after chronic hypoxia, likely decreased the stimulus for RV hypertrophy in cPLA_2α–/– mice. In addition, cPLA₂ deficiency may have attenuated RV hypertrophy by reducing the degree of polycythemia (18).

Since cPLA_2α–/– mice unexpectedly developed pulmonary vascular remodeling after prolonged breathing at F_IO₂ 0.10, we investigated whether prolonged hypoxic exposure restored HPV in cPLA_2α–/– mice. In wild-type mice, prolonged breathing at F_IO₂ 0.10 did not alter the pulmonary vasoconstrictor response to LMBO. This finding in mice contrasts with previous reports suggesting that chronic hypoxia attenuates HPV in rats (29) and is possibly attributable to differences in the methods used to monitor HPV (30). In cPLA_2α–/– mice, 3 weeks of breathing at F_IO₂ 0.10 restored the ability of LMBO to increase LPVR to the levels measured in wild-type mice without augmenting pulmonary PLA₂ activities (Figure 5). It is possible that currently unappreciated compensatory mechanisms provided AA in the cPLA_2α–/– mice after breathing at F_IO₂ 0.10 for 3 weeks, thereby restoring HPV. For example, compensatory downregulation of AA turnover in cellular membranes could lead to increased levels of AA in the cellular compartment (31). Alternatively, chronic hypoxia may increase basal pulmonary vascular tone by modifying pulmonary arterial myocyte function, augmenting synthesis of other vasoactive factors or enhancing vasoresponsiveness to these mediators (32), and thereby restored HPV in cPLA_2α–/– mice.

Recently, it has been suggested that inhibition of cPLA₂ may be beneficial in the treatment of acute lung injury. Nagase et al. reported that after administration of either lipopolysaccharide/zymosan or HCl, cPLA_2α–/– mice developed less lung injury, as reflected by improved oxygenation and decreased edema formation and polymorphonuclear neutrophil sequestration (33). These results suggest that from the perspective of systemic oxygenation in lung injury the beneficial anti-inflammatory effects of decreased cPLA₂ activity may be greater than the detrimental effects of cPLA₂ deficiency on HPV and that inhibition of cPLA₂-initiated pathways could be a promising strategy in the management of acute lung injury (20, 33). Nonetheless, our findings suggest that pharmacological inhibition of cPLA₂ should be approached with caution since the loss of cPLA₂ activity may impair the ability of the lung to match ventilation and perfusion and thereby cause systemic hypoxemia.

In summary, our studies revealed that a deficiency of cPLA₂ activity, induced either by gene deletion or by inhibition of this enzyme, abolished HPV in mice. These results also demonstrate that cPLA₂ is not essential for HPV in conditions of enhanced pulmonary vasoconstrictor tone. These results underscore an important modulatory role of cPLA₂ in HPV.

The authors thank Clary B. Clish for initial MS-MS analysis, Gudrun Mirick for technical assistance, Yuchiao Chang for statistical advice, and Edward A. Dennis for advice on the phospholipase activity assay. This study was supported in part by NIH grants HL-42397 (W.M. Zapol), HL-57172 (K.D. Bloch), DK-02493 (A. Sapirstein), DK-50305 (C.N. Serhan), and P01-DE-13499 (C.N. Serhan). R. Ullrich was supported by a grant from the Max Kade Foundation. K.D. Bloch was supported by an Established Investigator Award of the American Heart Association.

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