Role of 5,6-epoxyeicosatrienoic acid in the regulation of newborn piglet pulmonary vascular tone

Mamta Fuloria, Thuy K. Smith, and Judy L. Aschner

Department of Pediatrics, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We examined the responses of newborn piglet pulmonary resistance arteries (PRAs) to 5,6-epoxyeicosatrienoic acid (5,6-EET), a cytochrome P-450 metabolite of arachidonic acid. In PRAs preconstricted with a thromboxane A2 mimetic, 5,6-EET caused a concentration-dependent dilation. This dilation was partially inhibited by the combination of charybdotoxin (CTX) and apamin, inhibitors of large and small conductance calcium-dependent potassium (KCa) channels, and was abolished by depolarization of vascular smooth muscle with KCl. Disruption of the endothelium significantly attenuated the dilation, suggesting involvement of one or more endothelium-derived vasodilator pathways in this response. The dilation was partially inhibited by nitro-L-arginine (L-NA), an inhibitor of nitric oxide synthase (NOS), but was unaffected by indomethacin, a cyclooxygenase (COX) inhibitor. The combined inhibition of NOS and KCa channels with L-NA, CTX, and apamin abolished 5,6-EET-mediated dilation. Similarly, combined inhibition of NOS and COX abolished the response. We conclude that 5,6-EET is a potent vasodilator in newborn piglet PRAs. This dilation is mediated by redundant pathways that include release of nitric oxide (NO) and COX metabolites and activation of KCa channels. The endothelium dependence of this response suggests that 5,6-EET is not itself an endothelium-derived hyperpolarizing factor (EDHF) but may induce the release of one or more endothelium-derived relaxing factors, such as NO and/or EDHF.

pulmonary resistance arteries; calcium-dependent potassium channels; endothelium-derived hyperpolarizing factor; nitric oxide


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ARACHIDONIC ACID (AA) is metabolized to vasoactive products via the cyclooxygenase (COX), lipoxygenase, and cytochrome P-450 (cP450) monooxygenase pathways. The cP450 metabolism of AA results in the formation of a series of regiospecific and stereospecific epoxides [5,6-, 8,9-, 11,12-, and 14,15- epoxyeicosatrienoic acids (EETs)] and midchain and omega -terminal hydroxyeicosatetraenoic acids (HETEs) (19).

EETs are important regulators of systemic vascular tone and have been identified as a potential endothelium-derived hyperpolarizing factor (EDHF) in at least some vascular beds (7, 18, 21). EDHF is a factor(s) derived from the endothelium that mediates vasodilation by hyperpolarizing vascular smooth muscle (VSM) via activation of calcium-dependent potassium (KCa) channels. EDHF-mediated dilations and hyperpolarizations are insensitive to inhibitors of COX, nitric oxide synthase (NOS), and ATP-dependent K+ channels but are inhibited by the combination of charybdotoxin (CTX) and apamin, inhibitors of large, intermediate, and small conductance KCa channels (1, 10). EETs have been shown to mediate vasodilation in intestinal microvessels (23), caudal (8), cerebral (11, 17), renal (34), and coronary arteries (21, 26, 27). Various signaling pathways have been implicated in the dilation response to EETs. In some vascular beds, EET-induced dilation has been attributed to activation of KCa channels in VSM cells (12, 21). Others have demonstrated a COX-dependent (17, 29) or NO-dependent mechanism (30) in EET-mediated vasodilation.

EETs are endogenous products of rabbit (32), guinea pig (15), human, and rat lungs (31). However, there is a paucity of data on the role of EETs in the modulation of pulmonary vascular tone. All of the studies published to date have been performed in adult animals and report contradictory results (28-30, 33). Thus the role of EETs in the modulation of pulmonary vascular tone, especially in the newborn pulmonary circulation, has not been adequately addressed.

These studies were designed to test the hypothesis that 5,6-EET, the regioisomer shown to be most potent in the newborn piglet cerebral circulation (17), is an EDHF in the newborn piglet pulmonary microcirculation. Therefore, the aims of these studies were to 1) demonstrate that 5,6-EET is a dilator stimulus in the newborn piglet pulmonary microcirculation and 2) investigate the signaling mechanism(s) mediating 5,6-EET-induced pulmonary vasodilation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents

9,11-Dideoxy-11alpha ,9alpha -epoxymethanoprostaglandin F2alpha (U-46619) was purchased from Calbiochem (San Diego, CA). 5,6-EET, dissolved in acetonitrile, was purchased from Cayman Chemical (Ann Arbor, MI). All other chemicals were obtained from Sigma (St. Louis, MO). 17-Octadecynoic acid (17-ODYA) and miconazole were dissolved in dimethyl sulfoxide (DMSO).

5,6-EET, an intrinsically unstable compound that is readily oxidized upon contact with air, was stored at -80°F under argon or nitrogen. Working concentrations were prepared daily from the stock solution, kept on ice, and protected from light throughout the study protocol.

Animals

This study was approved by the Animal Care and Use Committee at Wake Forest University School of Medicine. Newborn piglets were housed in the Wake Forest University School of Medicine Animal Resource Facilities. This facility is maintained by the Department of Comparative Medicine and is fully accredited by the American Association for Accreditation of Laboratory Animal Care.

Isolation of Pulmonary Resistance Arteries

The heart and lungs were removed en bloc from newborn piglets (1-4 days old) after death with an overdose of pentobarbital sodium (75 mg/kg ip). Pulmonary resistance arteries (PRAs) with internal diameter of 150-300 µm and branching order of 9-12 (4) were dissected in Krebs-Henseleit buffer (118 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4 · 7H2O, 1.2 mM KH2PO4, 25 mM NaHCO3, 11 mM dextrose, and 2 mM CaCl2 · 2H2O). Side branches were tied with a single fiber of 10-0 multifilament braided nylon thread. Subsequently, the arteries were transferred to a well of an arteriograph (Living Systems Instrumentation, Burlington, VT) equipped with a heat controller that provides accurate control of the bath solution temperature. The chamber contains two microcannulas mounted on bulkheads to facilitate positioning. PRAs were cannulated at one end, secured with a single strand of suture, gently flushed free of blood with Krebs buffer, and cannulated at the distal end. The arteriograph was set in a 96-well plate holder on the stage of an inverted microscope (Nikon TMS) with a video camera attached to the viewing tube (Sony XC 73). A pressure servo system connected to the proximal cannula maintained intraluminal pressure at 15 ± 1 mmHg. The vessel image was projected on a television monitor, and the lumen diameter (LD) was continuously measured by a video dimension analysis system (Living Systems Instrumentation).

Experimental Protocol

Before the start of an experiment, the Krebs buffer in the vessel chamber was equilibrated with normoxic (21% O2) gas containing 5% CO2-balance N2 (pH 7.4). After a 30-min equilibration period with a stable baseline LD, the PRAs were constricted by addition of 50 mM KCl, followed by addition of acetylcholine (ACh; 10-5 M) or A-23187 (10-5 M), a calcium ionophore, to demonstrate intact VSM and endothelial function, respectively. Preparations that failed to constrict to KCl and/or failed to dilate to either ACh or A-23187 were excluded from further study. After another 30-min equilibration period with a stable baseline LD, the PRAs were constricted to ~30-45% of their resting diameter with U-46619 (10-7 M), a thromboxane A2 mimetic. Separate PRAs were used to assess the cumulative, concentration-dependent 5,6-EET-mediated responses under control conditions and in the presence of pharmacological inhibitors of various signaling pathways. At the end of each study, vascular integrity was assessed by demonstrating constriction to KCl (120 mM) and relaxation to sodium nitroprusside (SNP) and/or papaverine, followed by perfusion with calcium-free buffer to achieve a maximum LD. PRAs that failed to constrict to KCl and to dilate in response to either SNP or papaverine were excluded from analysis.

Specific Protocols

Protocol 1: response of preconstricted PRAs to 5,6-EET. After constriction to ~30-45% of resting LD with U-46619 (10-7 M), the cumulative responses of PRAs to increasing concentrations of 5,6-EET (10-9-10-5 M) were assessed.

Protocol 2: role of K+ channels in 5,6-EET-mediated dilation. We evaluated the role of membrane depolarization on 5,6-EET-mediated dilation by constricting PRAs with 80 mM KCl (instead of with U-46619). To determine the role of KCa channel activation, we pretreated PRAs for 30 min with the combination of CTX (5 × 10-8 M) and apamin (10-6 M), after which a new stable LD was determined. CTX is an inhibitor of large and intermediate conductance KCa channels; apamin inhibits small conductance KCa channels. The combination of CTX and apamin was used because this combination has been shown to inhibit EDHF-mediated dilations (1, 10).

Protocol 3: role of the endothelium in 5,6-EET-mediated dilation. To evaluate the endothelial-dependency of 5,6-EET-mediated dilation, we destroyed the endothelium of some PRAs by intraluminal perfusion of 2-10 ml of air, followed by a 10-min perfusion with Krebs buffer. Only air-denuded vessels that either failed to dilate or constricted in response to ACh or A-23187 were studied. After removal of the endothelium, the PRAs were constricted to ~30-45% of resting LD with U-46619 (10-7 M), and the responses to increasing concentrations of 5,6-EET (10-9-10-5 M) were assessed. At the end of each study, the denuded PRAs were exposed to KCl (120 mM) followed by SNP (10-5 M) and/or papaverine (10-4 M) to verify preservation of VSM function. In a separate series of 24 endothelium-denuded PRAs, we consistently observed a dilation response to SNP (10-5 M) at the end of the experiment, with a mean dilation of 51.7 ± 4.8%, demonstrating that VSM function is preserved after our denudation method.

Protocol 4: role of NO and COX metabolites in 5,6-EET-mediated dilation. We pretreated PRAs with nitro-L-arginine (L-NA, 10-4 M) for 30 min to assess the role of NO production in the dilation response to 5,6-EET. To determine whether products of the COX pathway were involved in 5,6-EET-mediated dilation, we used indomethacin (10-5 M × 30 min) to inhibit this pathway.

Protocol 5: role of cP450 metabolites in 5,6-EET-mediated dilation. To assess the role of cP450 metabolites in 5,6-EET-mediated dilation, we pretreated PRAs for 30 min with either 17-ODYA (5 × 10-5 M), a suicide substrate inhibitor of cP450 enzymes, or miconazole (2 × 10-5 M), an inhibitor of epoxygenase activity (25, 35).

Data Analysis

Unless otherwise indicated, n represents both the number of piglets and the number of PRAs studied. Data are expressed as mean ± SE. Statistical analysis was performed with SPSS 10.0. Repeated measure models were constructed with both within-group and between-group contrasts. Comparisons between groups were analyzed with a Bonferroni post hoc multiple-comparison test at the 0.05 level of significance.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

As shown in Fig. 1, PRAs preconstricted with U-46619 (10-7 M) demonstrated concentration-dependent dilation in the presence of 5,6-EET (10-9-10-5 M), with 62 ± 10% dilation observed at a concentration of 10-5 M. The dilation response to 5,6-EET was both rapid (within 90 s) and stable, resulting in dilation that was sustained for at least 10 min. In contrast, the vehicle control, acetonitrile, resulted in slight constriction (P = 0.007).


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Fig. 1.   Concentration-dependent dilation responses to 5,6-epoxyeicosatrienoic acid (5,6-EET) or vehicle control (acetonitrile) in U-46619-preconstricted pulmonary resistance arteries (PRAs; *P = 0.007 different from vehicle control).

Depolarization of VSM with KCl abolished 5,6-EET-mediated dilation (P = 0.002) (Fig. 2). The combination of CTX (5 × 10-8 M) and apamin (10-6 M) decreased baseline LD by 16 ± 2% (data not shown) and significantly inhibited 5,6-EET-mediated dilation (P = 0.02) (Fig. 2). These results suggest that activation of KCa channels and membrane hyperpolarization are involved in 5,6-EET-mediated dilation of neonatal piglet PRAs. These findings are also consistent with the involvement of an EDHF in 5,6-EET-mediated dilation response.


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Fig. 2.   Concentration-dependent dilation responses to 5,6-EET in U-46619-constricted PRA in the absence (control) and presence of apamin and charybdotoxin (CTX) and in PRAs constricted with KCl (*P < 0.05 for both treatment groups vs. control).

If 5,6-EET is an EDHF, then its exogenous addition should induce dilation independent of the endothelium. However, we found that removal/disruption of the endothelium significantly attenuated 5,6-EET-mediated dilation (P = 0.018) (Fig. 3). We therefore investigated the role of other endothelium-derived vasodilators in 5,6-EET-mediated dilation. Inhibition of NOS by L-NA (10-4 M) decreased baseline LD by 12 ± 4% and significantly inhibited 5,6-EET-mediated dilation (P = 0.005; Fig. 4). Addition of indomethacin (10-5 M) alone to inhibit the COX pathway had no effect on baseline tone or on 5,6-EET-mediated dilation (Fig. 4). As inhibition of NOS and of KCa channels both partially inhibited the dilation response, we examined 5,6-EET-mediated dilation in the presence of all three agents, L-NA, CTX, and apamin. The combination of L-NA, CTX, and apamin decreased baseline LD by 27 ± 4% and abolished 5,6-EET-mediated dilation (P < 0.05; Fig. 5). The combination of indomethacin and L-NA decreased baseline LD by 20 ± 7% and also abolished 5,6-EET-mediated dilation (P = 0.02; Fig. 5).


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Fig. 3.   Concentration-dependent dilation responses to 5,6-EET in endothelium-intact and disrupted PRAs (*P = 0.018 vs. endothelium-intact vessels).



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Fig. 4.   Concentration-dependent dilation responses to 5,6-EET in the absence (control) and presence of nitro-L-arginine (L-NA) or indomethacin (*P = 0.005 for L-NA-treated group vs. control; P = NS for indomethacin-treated group vs. control).



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Fig. 5.   Concentration-dependent dilation responses to 5,6-EET in the absence (control) and presence of L-NA, CTX, and apamin (Apa), and L-NA and indomethacin (Indo; *P < 0.05 for both treatment groups vs. control).

Inhibition of cP450 monoxygenases by either 17-ODYA (5 × 10-5 M), a specific cP450 suicide substrate inhibitor, or miconazole (2 × 10-5 M), an inhibitor of epoxygenase activity, decreased baseline LD by 7 ± 1 and 9 ± 4%, respectively, but had no effect on 5,6-EET-mediated dilation (data not shown). DMSO, the solvent for both 17-ODYA and miconazole, had no effect on baseline PRA diameter.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

EETs have been shown to be potent vasodilators in multiple circulatory beds and have been identified as potential EDHFs in the coronary and renal circulations (7, 18, 21). However, their role in the modulation of pulmonary vascular tone in the newborn remains unexplored. Therefore, we examined the effect of 5,6-EET in the neonatal piglet pulmonary microcirculation and designed studies to test the hypothesis that 5,6-EET is an EDHF in this circulatory bed.

The current study demonstrates that 5,6-EET is an endothelium-dependent vasodilator of neonatal piglet PRAs. 5,6-EET-mediated dilation is abolished by depolarization of VSM with KCl and is partially inhibited by the combination of CTX and apamin, inhibitors of large, intermediate, and small conductance KCa channels. These findings suggest that 5,6-EET mediates an EDHF-like vasodilation in the newborn piglet pulmonary microcirculation. 5,6-EET may directly or indirectly activate potassium channels with subsequent hyperpolarization of the VSM. Hyperpolarization of the VSM mediates vasodilation by closing voltage-gated calcium channels and reducing VSM intracellular calcium. Additionally, EETs have previously been shown to hyperpolarize endothelial cells by opening endothelial KCa channels (2). Endothelial cell hyperpolarization would cause an influx of calcium down its electrochemical gradient (endothelial cells lack voltage-dependent calcium channels). The subsequent increase in endothelial intracellular calcium would activate calcium-dependent enzymes, including endothelial NOS, thus stimulating a second indirect vasodilatory pathway.

If 5,6-EET is an EDHF, removal or disruption of the endothelium should not affect EET-mediated dilation when exogenously added. However, a significant finding of this study is that 5,6-EET-mediated vasodilation in the newborn piglet pulmonary microcirculation is significantly attenuated by removal of the endothelium. This finding refutes our hypothesis that 5,6-EET is, itself, an EDHF in the newborn piglet pulmonary microcirculation. The endothelium dependency of this dilation response in the newborn pulmonary circulation is in contrast to the findings of other investigators (13, 21) who have shown that endothelial denudation of porcine and canine coronary arteries does not alter the vasodilatory response to EETs. Similar to our findings, 5,6-EET-mediated vasodilation is an endothelium-dependent phenomenon in rat hepatic arteries (36) and in the rabbit superior mesenteric artery (14). The endothelium dependency of the dilation response observed by us can be interpreted in two ways. First, 5,6-EET is not itself an EDHF but may induce the release of an endothelium-derived relaxing factor, such as EDHF and/or endothelium-derived NO. Because two structurally and mechanistically different inhibitors of cP450 enzymes (miconazole and 17-ODYA) did not affect 5,6-EET-mediated dilation, the endothelium-derived vasodilators do not appear to be metabolites of the cP450 pathway. An alternative explanation is that intact myoendothelial gap junctions are required for 5,6-EET-induced dilation. Communication through gap junctions may permit heterocellular movement of ions and other small molecules and transmission of electrical stimuli (3), thus facilitating vascular responses. One possibility is that 5,6-EET hyperpolarizes endothelial cells and this hyperpolarization is then transmitted by electrogenic coupling to the VSM cells. In addition, there may be release of a freely diffusible factor from the endothelium (such as NO and/or EDHF) that may be essential for amplifying or propagating this local smooth muscle hyperpolarization. If such were the case, disruption/removal of the endothelium would interrupt both the transmission of the hyperpolarization and the endothelial release of factor(s) necessary for maintaining the hyperpolarization, thus attenuating the dilation response. Hutcheson and colleagues (14) have reported endothelium-dependent, NO-mediated dilation to 5,6-EET in the rabbit superior mesenteric artery. The NO-independent component of the response was abolished by Gap 27 peptide, an inhibitor of gap junction coupling, indicating a role for gap junctions in this dilation response.

Because 5,6-EET-mediated dilation of the pulmonary microcirculation is endothelium dependent, we investigated the role of NO, an endothelium-derived vasodilator, in this dilation response. We found that 5,6-EET-mediated dilation is significantly attenuated, but not abolished, by inhibition of NOS by L-NA. As shown in Fig. 4, at higher concentrations 5,6-EET is able to stimulate NO-independent dilation. This NO-insensitive dilation is blocked by KCa channel inhibition with CTX and apamin (shown in Fig. 5). NO has been shown to mediate dilation by a guanosine 3',5'-cyclic monophosphate-dependent mechanism or by direct activation of K+ channels (5). The additive effects of NOS inhibition and KCa channel inhibition suggest that NO mediates dilation by a mechanism independent of KCa channel activation. This finding is consistent with our preliminary data in PRA, demonstrating that inhibition of KCa channels with CTX has no effect on the dilation response to the NO donor S-nitroso-N-acetylpenicillamine.

Inhibition of the NO-insensitive dilation response to 5,6-EET by CTX and apamin requires further discussion in light of the endothelium dependency of the response. Neither K+ channel blocker is site specific; both are capable of inhibiting KCa channels on the endothelium, the VSM, or both. Our findings are consistent with 5,6-EET activating KCa channels on the endothelium, either directly or indirectly via release of another endothelium-derived hyperpolarizing factor. Our finding that the combination of L-NA and indomethacin also abolishes the response suggests that the NO-insensitive dilation response may be caused by 5,6-EET-induced release of a COX metabolite that can hyperpolarize the VSM. What is clear from our data is that 5,6-EET does not directly activate VSM KCa channels on PRA because the response is endothelium dependent.

Our data demonstrating that 5,6-EET-mediated dilation in PRA was predominantly NO dependent are similar to findings by Tan et al. (30) in isolated rabbit lungs with elevated pulmonary vascular tone. The small NO-independent component of this response was mediated via activation of COX (30). It is possible that the dominant pathway involved in 5,6-EET-mediated dilation of the neonatal pulmonary microcirculation is the production of NO. As previously described by Cohen et al. (9), L-NA alone may not entirely prevent the release of all NO, raising the possibility that an L-NA-noninhibitable pool of NO may mediate the residual vasodilatory response. Alternatively, NO stored in the vascular wall in protein-bound dinitrosyl-iron complexes may generate low-molecular-weight dinitrosyl-iron complexes, which can activate soluble guanylate cyclase and promote vascular relaxation (20).

Inhibition of COX by itself did not affect 5,6-EET-mediated dilation of piglet PRAs. However, the combined inhibition of NOS and COX with L-NA and indomethacin, respectively, abolished 5,6-EET-mediated dilation. This finding suggests that COX metabolites play a secondary role in 5,6-EET-mediated dilation of the pulmonary microcirculation; inhibition of NO synthesis seems to be required to uncover the operation of this vasodilatory pathway. As has previously been reported (6), reduced prostacyclin levels after inhibition of COX with indomethacin may result in increased NO production, which might compensate for the lack of prostacyclin generation. Similar to our findings, Stephenson and colleagues (29) have shown 5,6-EET-induced, COX-dependent vasodilation in prostaglandin F2alpha -constricted isolated canine pulmonary venous rings. In isolated perfused canine lungs preconstricted with U-46619, 5,6-EET caused a COX-dependent decrease in pulmonary vascular resistance secondary to changes in resistance in the pulmonary venous segment. In contrast, when 5-hydroxytryptamine was used as the constrictor agent, 5,6-EET caused a decrease in pulmonary vascular resistance by decreasing pulmonary arterial resistance. The authors concluded that 5,6-EET-induced vasodilation depended on the constrictor agent present, the segmental resistance, and COX activity (28). In contrast to all other reported findings, including our own, Zhu and colleagues (33) have reported 5,6-EET-mediated, endothelium- and COX-dependent vasoconstriction in isolated, pressurized, rabbit pulmonary arteries, possibly due to formation of a pressor prostanoid compound. The vasoconstriction could be blocked by pretreatment with a thromboxane receptor antagonist. The differences in the signaling mechanisms observed by investigators may be related to interspecies differences, age of the animals studied, size of the vessels studied (resistance vs. conductance vessels), and/or the preparation studied (vessel rings, isolated perfused lungs vs. cannulated vessels).

We have observed that inhibition of only one dilatory pathway significantly inhibits, but does not abolish, 5,6-EET-mediated vasodilation of newborn piglet PRAs. Inhibition of multiple pathways is required to abolish 5,6-EET-mediated vasodilation. Thus 5,6-EET-induced vasodilation in newborn piglet PRAs is mediated by redundant mechanisms that include production of NO and COX metabolites and activation of KCa channels. The redundancy of these signaling mechanisms is not unique to 5,6-EET, as modulation of vascular tone, particularly in the resistance circulation, rarely depends entirely on the integrity of a single signaling pathway. The involvement of multiple signaling molecules in this dilation response demonstrates the built-in complexity of the signaling mechanisms that modulate pulmonary vascular tone and the potential protection afforded by this duplication and redundancy of pathways. Intercommunication between various signaling pathways provides a backup mechanism whereby compromise of one signaling pathway may be compensated for by other intact mechanisms.

Although our study is limited to the responses of PRA in vitro to exogenous EET administration, it does establish that newborn PRAs possess the downstream signaling pathways that are required for a dilation response upon exposure to exogenous EETs. Although it has been shown that EETs are endogenous products of mature rabbit (32), guinea pig (15), human, and rat lungs (31), the capacity for endogenous EET production in newborn lungs from humans or piglets has not been unequivocally established. Our findings that both 17-ODYA and miconazole cause constriction of PRA at baseline tone are indirect evidence that endogenously produced cP450 metabolites modulate resting tone in newborn pulmonary resistance vessels. Unfortunately, these cP450 inhibitors are not totally specific, limiting conclusions about the cP450 isoform(s) involved or the active metabolite produced. For example, miconazole, an inhibitor of epoxygenase activity, also binds to heme and inhibits enzymes involved in drug metabolism, steroidogenesis, and the synthesis of NO (25). The anitmycotic agents also alter intracellular calcium concentrations and ion channel activity in some cell types (25). 17-ODYA, a fairly specific suicide substrate inhibitor, will effectively block the formation of 20-HETE in addition to the EETs. However, because 20-HETE is a vasoconstrictor in most vascular beds, it is reasonable to conclude that the decrease in LD observed with 17-ODYA reflects blockade of an endogenous cP450-derived dilator, presumably an EET. Unpublished data from our laboratory demonstrate that AA (10-9-10-5 M), but not the vehicle control ethanol, induces a 33 ± 6% dilation at a concentration of 10-5 M (n = 4) in the presence of indomethacin to block the production of dilator prostanoids via the COX pathway of AA metabolism. This provides further indirect support for endogenous production of EETs in newborn PRAs. Further studies are needed to determine the metabolic capacity of the newborn lung to produce various regio- and steriospecific EET isoforms and whether the cP450 isoforms present in the lung are developmentally regulated, as has been demonstrated in the kidney (16, 22).

In summary, this study demonstrates that 5,6-EET is a vasodilator of the newborn piglet pulmonary microcirculation. An important finding of this study is that, unlike many systemic circulatory beds, the dilation response of the newborn piglet pulmonary circulation is endothelium dependent, suggesting that 5,6-EET induces release of one or more endothelium-derived relaxing factors. Alternately, intact myoendothelial gap junctions may be required for 5,6-EET-mediated vasodilation. Mechanisms involved in 5,6-EET-mediated vasodilation include the release of NO and COX metabolites and activation of KCa channels. The role of COX metabolites in 5,6-EET-mediated dilation was observed only when the NO pathway was compromised, suggesting that endogenous NO may modulate prostanoid production in the newborn pulmonary circulation. We speculate that, in addition to NO and prostacyclins, products of the cP450 pathway of AA metabolism (i.e., the EETs) may play an important role in the regulation of neonatal pulmonary vascular tone.


    ACKNOWLEDGEMENTS

This work was supported by the Wake Forest University School of Medicine Intramural Grant program (to M. Fuloria) and by National Heart, Lung, and Blood Institute Grant HL-62489 (to J. L. Aschner).


    FOOTNOTES

Address for reprint requests and other correspondence: M. Fuloria, Dept. of Pediatrics, Wake Forest Univ. School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157 (E-mail: mfuloria{at}wfubmc.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.

March 29, 2002;10.1152/ajplung.00444.2001

Received 16 November 2001; accepted in final form 24 March 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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