Role of EETs in regulation of endothelial permeability in rat lung

Diego F. Alvarez, Eli-Anne B. Gjerde, and Mary I. Townsley

Department of Physiology, University of South Alabama, Mobile, Alabama 36688

Submitted 13 May 2003 ; accepted in final form 17 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study tested the hypothesis that epoxyeicosatrienoic acids (EETs) derived from arachidonic acid via P-450 epoxygenases are soluble factors linking depletion of endoplasmic reticulum Ca2+ stores and store-dependent regulation of endothelial cell (EC) permeability in rat lung. EC permeability was measured via the capillary filtration coefficient (Kf,c) in isolated, perfused rat lungs. 14,15-EET and 5,6-EET increased EC permeability, a response that was significantly different from that of 8,9-EET, 11,12-EET, and vehicle control. The permeability response to 14,15-EET was not significantly attenuated by the nonspecific Ca2+ channel blocker Gd3+ (P = 0.068). In lungs perfused with low [Ca2+], 14,15-EET tended to increase EC permeability, although a significant increase in Kf,c was observed only following Ca2+ add-back. As positive control, we showed that the 3.7-fold increase in Kf,c evoked by thapsigargin (TG), a known activator of store depletion-induced Ca2+ entry, was blocked by both Gd3+ and low [Ca2+] buffer. Nonetheless, the permeability response to TG could not be blocked by the phospholipase A2 inhibitors mepacrine or methyl arachidonyl fluorophosphonate or the P-450 epoxygenase inhibitors 17-octadecynoic acid or propargyloxyphenyl hexanoic acid. Similarly, combined pretreatment with ibuprofen and dicyclohexylurea to block EET metabolism had no effect on the permeability response to TG. We conclude that EETs have a heterogeneous impact on EC permeability. Despite a requirement for Ca2+ entry with both TG and 14,15-EET, our data suggest that distinct signaling pathways or heterogeneity in EC responsiveness is responsible for the observed EC injury evoked by EETs and store depletion in the isolated rat lung.

capillary filtration coefficient; store depletion; epoxyeicosatrienoic acid; cytochrome P-450


CALCIUM (Ca2+) entry into pulmonary endothelial cells (EC) is well recognized to promote gap formation and increase EC permeability, although the Ca2+ channels involved and the interplay between signaling mechanisms regulating this process have not been as well delineated (22, 39). Numerous studies have demonstrated that regulation of Ca2+ entry in EC is often linked to depletion of the endoplasmic reticulum Ca2+ store, which subsequently leads to Ca2+ entry (4, 22), a process known as capacitative Ca2+ entry (28). The link between store depletion and capacitative Ca2+ entry has been thought to depend on either soluble mediators or physical coupling between the endoplasmic reticulum and plasma membrane Ca2+ channel(s). Although the notion that physical coupling mechanisms are involved in EC has gained support (26, 47), a link involving soluble mediators has not been ruled out. Among the candidates proposed for the latter are arachidonic acid (AA) metabolites generated via cytochrome P-450 epoxygenases (epoxyeicosatrienoic acids or EETs). EETs have been implicated in regulation of capacitative Ca2+ entry in a number of cell types, including thymocytes, astrocytes, and systemic EC (1, 13, 33).

Involvement of P-450 epoxygenase metabolites in regulation of Ca2+ entry and EC permeability has been previously proposed. In the canine lung, we have shown that the store depletion-induced increase in lung EC permeability can be completely blocked by using 17-octadecynoic acid (17-ODYA) and clotrimazole to inhibit P-450 epoxygenases (18), an observation that supports the notion that EETs play an important role in regulation of EC permeability. However, there is no evidence at this point that this regulatory mechanism is an obligate participant in regulation of EC permeability regardless of the source or the injury paradigm. Because cytochrome P-450 epoxygenases are known to be expressed in peripheral rat lung (32), we sought to evaluate their role in capacitative Ca2+ entry and increased EC permeability in this species. The specific goal of this study was to test the hypothesis that EETs are involved in the store-dependent regulation of EC permeability in rat lung.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Isolated Lung Preparation

The isolated lung preparation has been previously described (5, 23, 42). Adult male CD40 rats (n = 113, 368 ± 10 g, mean ± SE) were anesthetized with pentobarbital sodium (50 mg/kg ip). After tracheotomy, mechanical ventilation was initiated with room air at 50-70 strokes per min (6 ml/kg body wt). The thorax was opened via a subdiaphragmatic incision, and then 5,000 units of heparin were injected into the left ventricle and allowed to circulate for 5 min. A plastic cannula (PE 240) connected to a reservoir was advanced into the pulmonary artery via an incision in the right ventricular free wall, and the cannula was secured with 3-0 silk; low flow of perfusate was maintained. Next, a plastic cannula was advanced into the left atrium via an incision in the apex of the left ventricle. Both cannulas were secured by umbilical tapes tied around the ventricles. The lung and the heart were removed en bloc and suspended from a calibrated force transducer. The lung was ventilated (5% CO2, 30% O2, balance N2), perfused at constant flow (38°C) with Earle's buffer salt solution containing 4% albumin (unless indicated) and pH adjusted to 7.4; total circulating volume was ~45 ml. Initially in all experiments, an isogravimetric state was established with the lung perfused under zone 3 conditions. Venous pressure (Pv) was set to 4 cmH2O and positive end-expiratory pressure to 3 cmH2O. Arterial pressure (Pa), Pv, airway pressure, and lung weight were continuously recorded on an Astromed polygraph (model 7400).

Evaluation of EC Permeability and Hemodynamics

The capillary filtration coefficient (Kf,c) was measured as an index of pulmonary EC permeability (5, 42). Kf,c was determined by dividing the rate of lung weight gain measured 13-15 min after increasing Pv by 7-10 cmH2O by the resultant increment in capillary pressure (Pc) (43) and normalizing the result per gram dry lung weight. This rate of weight gain was corrected for any nonisogravimetric rate of weight gain during the period just preceding the Kf,c maneuver. Pc was measured by the double occlusion technique (41). Kf,c measurements were made at baseline and at specific time points (30-90 min) after treatment, as described under experimental protocols. Using measures of Pa, Pc, and Pv, we determined not only total vascular resistance (Rt) but also arterial (Ra) and venous (Rv) vascular resistance (41, 43).

Experimental Protocols

Time controls. To demonstrate the stability of the isolated lung preparation in our hands, lungs were isolated and perfused as described and flow increased to the maximal isogravimetric flow in zone 3. Baseline Kf,c and hemodynamics were measured initially, and then the lungs were subsequently perfused at a constant flow for ~3 h before final measurements of Kf,c and hemodynamics were repeated (n = 6).

Effect of EETs on lung EC permeability and hemodynamics. The epoxygenase metabolites of AA are synthesized as a family of four regioisomers, including 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET (32). EETs have been proposed as mediators of acute lung injury in canine lung, although only the effect of 5,6-EET has been reported (18, 38). We evaluated the effect of each EET regioisomer independently on Kf,c in the isolated rat lung. Vehicle (0.91 ml ethanol, n = 8) or EET (3 µM, n = 5-8) was added as a bolus to the venous reservoir following the baseline measurements. Final hemodynamics and Kf,c were measured after 60 min.

Requirement for Ca2+ entry. EETs have been associated with Ca2+ entry in aortic EC (16, 21) and Ca2+ entry in EC often results in increased permeability (17, 39). Thus in these protocols we tested whether the observed increase in Kf,c following 14,15-EET involved Ca2+ entry. After the baseline measurements, lungs were pretreated with gadolinium chloride (Gd3+), a lanthanide mineral that nonselectively inhibits cation channels and capacitative Ca2+ entry (44). Because of the ability of Gd3+ to bind to albumin (3), lungs used for these experiments were perfused throughout with 1% albumin-3% clinical grade dextran, a combination that does not alter Kf,c in the lung (30). The lungs were treated for 30 min with vehicle (50 µl H2O, n = 8) or Gd3+ (30 µM, n = 5) followed by administration of 14,15-EET (3 µM). Final measures of hemodynamics and Kf,c were made 60 min later. Thapsigargin (TG), an alkaloid derivative that blocks Ca2+ reuptake into the endoplasmic reticulum and evokes capacitative Ca2+ entry (40), was used as a positive control (4, 17). After baseline measurements, lungs were pretreated with vehicle (n = 6) or Gd3+ (n = 5) for 30 min before addition of TG (150 nM). After 60 min, the final measurements of hemodynamics and Kf,c were made. To further address the requirement for Ca2+ entry in 14,15-EET-induced lung injury, we perfused lungs with a bicarbonate-buffered physiological salt solution (in mM: 116.0 NaCl, 5.2 KCl, 0.9 MgSO4, 1.0 Na2HPO4, and 8.3 D-glucose) containing 4% bovine serum albumin and either physiological (2.2 mM) or low (0.02 mM) CaCl2 (4). After measurement of baseline hemodynamics and Kf,c, lungs were treated with 14,15-EET (3 µM, n = 5) for 45 min before a second Kf,c was made. Subsequently, CaCl2 was added to the perfusate to achieve a 2.2 mM circulating concentration, and the final Kf,c was measured 15 min later. In parallel experiments, we evaluated the effect of low Ca2+ and Ca2+ add-back on the permeability response to TG (150 nM, n = 5), as a positive control.

Dependence of TG-induced lung injury on EETs. EETs have been postulated to act as soluble factors involved in capacitative Ca2+ entry (1, 13). Thus we tested whether TG-induced EC injury in rat lung was dependent on EET synthesis, as previously shown for canine lung (18). First, the effect of TG alone was documented (150 nM, n = 12). Then, we evaluated whether EETs play a role in the response to TG by blocking sequential steps in EET synthesis and metabolism, starting with mobilization of the P-450 substrate AA by phospholipase A2 (PLA2). To block activation of PLA2, we utilized mepacrine (100 µM, n = 5), a nonspecific PLA2 inhibitor, or the more selective inhibitor methyl arachidonyl fluorophosphonate (MAFP, 2.5 µM, n = 4) (31, 48). In separate studies, we tested whether AA metabolism via the P-450 pathway was involved, using the P-450 inhibitor 17-ODYA (5 µM, n = 8) or the specific epoxygenase inhibitor propargyloxyphenyl hexanoic acid (PPOH, 50 µM, n = 9) (18, 46). To test whether EET metabolism by cyclooxygenase or epoxide hydrolase was a limiting factor, the cyclooxygenase inhibitor ibuprofen (30 µM) and the epoxide hydrolase inhibitor dicyclohexylurea (DCU, 3 µM) were added in combination (n = 5) (7, 50). For all experiments in this series, lungs were pretreated with the inhibitors after the baseline measurements and 30 min before addition of TG (150 nM). Final measurements of hemodynamics and Kf,c were made 60 min after addition of TG. To assure that the observed increases in EC permeability were not influenced by the inhibitors, we also tested each inhibitor in the absence of TG.

Drugs

EETs were obtained from Biomol. MAFP and PPOH were obtained from Cayman Chemical. KCl, CaCl2, and Na2HPO4 were obtained from Fisher. All other reagents were purchased from Sigma Chemical. TG was dissolved in DMSO, aliquoted, and stored protected from light at -20°C. Mepacrine and Gd3+ were dissolved in H2O and stored at room temperature. DCU was dissolved in H2O and stored protected from light at 4°C. 17-ODYA and ibuprofen were dissolved in 90% ethanol and stored at room temperature. Drugs were added as a bolus to the venous reservoir; concentrations noted are final concentrations in the perfusate. Bolus volumes for EETs (or EET vehicle) were 0.91 ml. For all other drugs, the bolus volume was <100 µl.

Statistics

Data are presented as means ± SE. Statistical comparisons between groups were done using one-way or two-way analysis of variance (ANOVA) with repeated measures followed by Tukey's or Bonferroni's post hoc t-test, respectively, as appropriate. P values < 0.05 were considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The stability of the isolated rat lung in our hands was confirmed by time control experiments. Using a perfusate flow of 21.8 ± 0.7 ml/min (~0.06 ml/g body wt) and Pv set to 4.0 cmH2O, baseline Pa and Pc averaged 21.3 ± 0.4 and 13.3 ± 0.3 cmH2O, respectively, in this group, and Rt averaged 0.79 ± 0.03 cmH2O·ml-1·min. None of these hemodynamic parameters was altered by 3-h perfusion, even at this flow rate. The baseline Kf,c was not different from the final Kf,c measured after 3 h of constant flow perfusion (0.0071 ± 0.0010 vs. 0.0081 ± 0.0010 ml·min-1·cmH2O-1·g dry weight-1, respectively). In the remaining protocols, lungs were perfused at 14-16 ml/min with either albumin-normal [Ca2+], albumin-dextran, or albumin-low [Ca2+]. When these groups overall were compared, the albumin-dextran perfusate was found to result in significant pulmonary hypertension, as evidenced by increases in Rt (1.44 ± 0.05 vs. 1.17 ± 0.03 cmH2O·ml-1·min, P < 0.05), Pa (27.2 ± 0.5 vs. 19.8 ± 0.3 cmH2O, P < 0.05), and Pc (18.0 ± 0.3 vs. 12.7 ± 0.2 cmH2O, P < 0.05) compared with lungs perfused with albumin-normal [Ca2+]. In contrast, the low [Ca2+] buffer decreased Rt (0.78 ± 0.02 vs. 1.17 ± 0.03 cmH2O·ml-1·min, P < 0.05), Pa (15.2 ± 0.2 vs. 19.8 ± 0.3 cmH20, P < 0.05), and Pc (9.0 ± 0.1 vs. 12.7 ± 0.2 cmH2O, P < 0.05), compared with that in lungs perfused with physiological [Ca2+]. Nonetheless, as shown in Table 1, there were no differences in the baseline Kf,c in the lungs perfused with albumin-dextran or albumin-low [Ca2+] compared with those perfused with albumin-normal [Ca2+].


View this table:
[in this window]
[in a new window]
 
Table 1. Baseline measurements in isolated rat lung

 

Effect of EETs on EC Permeability and Hemodynamics

To address whether EETs affect Kf,c in the isolated rat lung, we tested each of the four regioisomers. EETs had a heterogeneous effect on EC permeability. The results are shown in Fig. 1, where the paired permeability responses measured at baseline and after treatment with each of the four EET regioisomers are compared. There was a significant increase in EC permeability with 5,6-EET (2.6-fold) and 14,15-EET (4.2-fold) when the final Kf,c was compared relative to baseline. These responses were significantly different than those resulting from 8,9-EET, 11,12-EET, or vehicle control, based on two-way ANOVA with repeated measures. Although there were no differences in baseline hemodynamics between groups, treatment of the lungs with 11,12- and 14,15-EET resulted in a mild increase in Rt. 11,12-EET increased Rt from 1.51 ± 0.24 to 1.71 ± 0.31 cmH2O·ml-1·min (baseline vs. final, P < 0.05), whereas 14,15-EET increased Rt from 1.51 ± 0.14 to 1.66 ± 0.15 cmH2O·ml-1·min (baseline vs. final, P < 0.05). We did not observe this response in any other group (two-way ANOVA with repeated measures). Furthermore, the increase in Rt (baseline vs. final) primarily resulted from increased resistance in the arterial pulmonary vascular compartment for 11,12-EET (Ra increased from 0.65 ± 0.13 to 0.81 ± 0.21 cmH2O·ml-1·min), whereas the venous compartment was primarily targeted by 14,15-EET (Rv increased from 0.87 ± 0.12 to 0.99 ± 0.14 cmH2O·ml-1·min); P < 0.05 as compared by paired t-test.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1. Regiospecific effect of epoxyeicosatrienoic acids (EETs) on endothelial cell (EC) permeability. The EC permeability response evoked by each of the 4 EET regioisomers was evaluated in the isolated rat lung using measurement of capillary filtration coefficient (Kf,c) at baseline (BL, open bars) and the final measurement (F, closed bars) after treatment with EET (3 µM) or vehicle. The increase in EC permeability induced by 5,6- and 14,15-EET was significantly different compared with the other groups (*P < 0.05, 2-way ANOVA with repeated measures).

 

Role of Extracellular Ca2+ in the Permeability Response to EETs

To determine whether the observed EET-induced increase in EC permeability was due to Ca2+ entry, we evaluated the permeability response to 14,15-EET in the absence and presence of the nonselective cation channel blocker Gd3+ and compared it with the TG response. There was no difference in the baseline Kf,c in either group compared with lungs perfused with albumin alone (see Table 1). However, the response to 14,15-EET in the absence of Gd3+ was significantly less (P < 0.05) in the albumin-dextran perfused lungs (2.5-fold increase in Kf,c) compared with the response in lungs perfused with albumin alone (4.2-fold, P < 0.05), whereas the response to TG was unchanged. The permeability response to TG was significantly attenuated in the presence of 30 µM Gd3+, as shown in Fig. 2 (P < 0.05, two-way ANOVA), confirming that Ca2+ entry is required for the EC injury induced by TG. In contrast, the tendency for the response to 14,15-EET to be reduced by Gd3+ was not statistically significant (P = 0.068), suggesting that parallel mechanisms independent from Ca2+ may be involved. To more clearly elucidate a definitive role for Ca2+ in the EET-induced lung injury, we utilized a perfusate with minimal [Ca2+] (4). A dose-response curve was first generated to determine the lower limit for perfusate [Ca2+], which allowed stability of the lung for 2 h, as measured by Kf,c. These data show that Kf,c remained stable with a perfusate [Ca2+] as low as 0.02 mM (0.0142 ± 0.0010 vs. 0.0194 ± 0.0008 ml·min-1·cmH2O-1·g dry wt-1, baseline vs. final respectively, n = 3). Although baseline Kf,c remained normal at 0.01 mM [Ca2+], final Kf,c was elevated (data not shown). Thus we chose to set the low [Ca2+] at 0.02 mM for further study. The results (Fig. 3) show that in lungs treated with 14,15-EET, Kf,c tended to increase despite the low [Ca2+] (P = 0.09). Nonetheless, a significant increase in Kf,c was elicited upon Ca2+ add-back (P < 0.05 compared with baseline, as analyzed by one-way ANOVA). In contrast, TG-induced lung injury was prevented in the low [Ca2+] buffer, whereas addition of Ca2+ to achieve a physiological concentration of 2.2 mM restored the TG-induced increase in EC permeability (P < 0.05 vs. baseline and low [Ca2+], one-way ANOVA). These data provide support for the notion that 14,15-EET elicits an increase in lung EC permeability via Ca2+-dependent mechanisms but that the response likely involves Ca2+-independent mechanisms as well.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2. Effect of Ca2+ entry blockade on 14,15-EET and thapsigargin (TG)-induced increases in EC permeability. The lanthanide mineral Gd3+ was used as a nonselective Ca2+ channel blocker to evaluate a requirement for extracellular Ca2+ in the permeability response to 14,15-EET (3 µM) or TG (150 nM). Baseline Kf,c (BL, open bars) was compared with final Kf,c (F, closed bars) after treatment with either 14,15-EET or TG. Pretreatment with Gd3+ did not clearly attenuate the 14,15-EET response (P = 0.068), although Gd3+ did block the permeability response to TG (*P < 0.05 vs. BL, 2-way ANOVA).

 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3. Evaluation of Ca2+ entry in 14,15-EET-induced lung injury. Whether Ca2+ was an absolute requirement involved in EET-induced increases in Kf,c was established by using a low [Ca2+] buffer. In the presence of 0.02 mM [Ca2+], Kf,c was measured at baseline (BL, open bars) and after treatment with 14,15-EET or TG (striped bars). In the same experiments, Kf,c was again measured after Ca2+ add-back (2.2 mM [Ca2+], closed bars). 14,15-EET-induced injury was observed upon Ca2+ add-back, although the response was not significantly different compared with that in the presence of low [Ca2+]. Low [Ca2+] buffer blocked the TG-induced injury, a response that was reestablished after Ca2+ add-back. P < 0.05 vs. baseline (*) or agonist in low [Ca2+](**) within the same group (1-way ANOVA).

 

Involvement of EETs in Capacitative Ca2+ Entry

To evaluate the potential involvement of the P-450-mediated AA metabolism in the TG-induced increase in EC permeability, we used inhibitors to target several steps in the AA cascade. As shown in Fig. 4, TG increased EC permeability 3.7-fold (P < 0.05), a response that was not blocked by pretreatment of the lung with the PLA2 inhibitors mepacrine or MAFP. In contrast to our observation in the canine lung, inhibition of P-450 activity with either 17-ODYA or PPOH did not alter the TG-induced permeability response in the rat lung. Similarly, inhibition of EET metabolism with ibuprofen and DCU had no effect on the TG-induced increase in EC permeability. A further analysis showed no significant difference between groups, except for the mepacrine pretreatment, which enhanced the response to TG (P < 0.05, two-way ANOVA with repeated measures). Collectively, these results provide support for the notion that P-450 epoxygenase metabolites are not involved in the link between TG-induced store depletion and increased EC permeability in the rat lung. None of these inhibitors had any effect on Kf,c in the absence of TG (data not shown).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4. Role of EETs in the permeability response to TG. The requirement for EETs in the TG-induced increase in EC permeability was evaluated using inhibitors of the arachidonic acid metabolic cascade. Baseline Kf,c (BL, open bars) was compared with the final measurement (F, closed bars) after treatment with the different inhibitors in the presence of TG. TG (150 nM) evoked a significant permeability response (*P < 0.05 vs. BL, paired t-test) in the presence of phospholipase A2 (PLA2) inhibitors [mepacrine or methyl arachidonyl fluorophosphonate (MAFP)] or by P-450 inhibitors [17-octadecynoic acid (17-ODYA) or propargyloxyphenol hexanoic acid (PPOH)]. Similarly, the permeability response was maintained after combined treatment with ibuprofen and dicyclohexylurea (DCU) to block EET metabolism. There was no significant difference between groups, except for the enhanced response to TG observed following treatment with mepacrine (2-way ANOVA with repeated measures).

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study provides the first evidence that EETs regulate EC permeability in the rat lung in a regiospecific manner. Though the mechanism by which EETs increase permeability appears to involve Ca2+ entry, a parallel Ca2+-independent pathway also may contribute. The role of endogenous EETs in regulation of lung EC permeability is unclear. P-450 epoxygenases are known to be expressed in the lung. Specifically in rat lung, expression of epoxygenases from the CYP2C, 2B, 2J, and 1A families has been demonstrated (32). The EET dose chosen for this study (3 µM) is similar to the circulating concentration found in perfused human lung ex vivo, after challenge with Ca2+ ionophore (20). Although 5,6- and 14,15-EET clearly increase permeability, our results suggest that these P-450 metabolites are not involved in the link between store depletion and Ca2+ entry in rat lung EC.

One question that may arise when considering the variable response to the EET regioisomers is whether the lung preparation per se is stable. We found that rat lungs could be perfused at a higher flow (0.04-0.06 ml·min-1·g body wt-1) compared with that typically used (0.03 ml·min-1·g body wt-1) (4, 19), while still retaining an isogravimetric state at baseline. As a result, Pa and Pc were higher than those described at lower flow but were similar to those reported in vivo (34). In the time control experiments, we demonstrated that even a flow rate of 0.06 ml·min-1·g body wt-1 did not compromise the lung, as evidenced by maintenance of a constant Kf,c during the 3-h experiment. A benefit of higher flow is better recruitment of exchange area in the lung (35, 42). Given the stability of the preparation, we believe that the differing responses to the EET regioisomers was not due to deterioration in the lung preparation.

Our results showed heterogeneity in the effect of EET regioisomers on pulmonary EC permeability. Regiospecificity with respect to other actions of EETs is well recognized. EETs may relax or contract airway or vascular smooth muscle, depending on the tissue, isomer, and concentration. EETs have been found to exert vasoactive effects on the pulmonary vasculature in in vitro and in vivo studies. In isolated canine lung, 5,6-EET induced vasodilation that was localized to the arterial compartment (36). In vitro studies have shown contraction of pulmonary artery rings induced by all of the four EET regioisomers in rabbits (51) and rats (49). More recently, 5,6-EET was found to produce dilation of extralobar pulmonary artery rings, whereas vasoconstriction was observed in intrapulmonary arteries in isolated rabbit lung (37). Although not the primary focus of our study, our results indicate that there was a mild increase in the total resistance of pulmonary vessels following treatment with 11,12- and 14,15-EET, but not the other two regioisomers. Although these results support the notion that specific EETs are vasoactive, there are clearly regioisomer- and species-dependent differences in the magnitude, targeted vascular compartment, and directionality of that vasoactive effect. One concern that is often raised is whether increases in vascular resistance affect the measure of EC permeability. Previous studies have shown that Kf,c is unresponsive to even fourfold increases in Rt induced by vasoconstrictors (29). Furthermore, an impact of vasoconstriction can be ruled out in our studies since 11,12- and 14,15-EET both increased Rt, yet only 14,15-EET altered EC permeability.

EETs have been shown to promote Ca2+ entry in a number of cell types, including systemic EC (13, 16, 29). The mechanism by which these P-450-derived lipids promote Ca2+ entry may be multifactorial and tissue dependent. We have preliminary evidence supporting the notion that 14,15-EET promotes Ca2+ entry in rat lung microvascular EC (11). Although there is ample evidence that EETs promote Ca2+ entry in systemic EC, there have been no other reports in which their effect on Ca2+ entry in pulmonary EC has been studied. Because EETs were demonstrated in the present study to increase pulmonary EC permeability and since Ca2+ entry in EC often results in increased EC permeability (4, 9, 17, 26), it was reasonable to postulate that EET-induced Ca2+ entry explained the resultant increase in Kf,c observed in this study. We used two different approaches to address this hypothesis: the lanthanide mineral Gd3+, used as a nonspecific cation channel blocker, and a low [Ca2+] perfusate, used to directly test the requirement for Ca2+ entry. Gd3+ blocks store depletion-induced Ca2+ entry via transient receptor potential channels, which have been characterized as putative store-operated channels (2, 14, 25). Gd3+ is difficult to use in vivo since it binds avidly to albumin (3), which may limit its efficacy with respect to blockade of Ca2+ channels. By using a combination of 1% albumin with 3% dextran, we could minimize binding of Gd3+ to albumin yet maintain adequate colloid in the perfusate and normal baseline permeability (30). The observation that 14,15-EET was less effective in lungs perfused with the albumin-dextran mixture was surprising, particularly since the response to TG was unchanged. This could potentially be due to increased EET metabolism, based on the decreased albumin binding capacity in this perfusate. To identify the optimal Gd3+ concentration, we completed a preliminary dose-response relationship, using the degree of inhibition of the TG-induced increase in Kf,c with Gd3+ pretreatment as our endpoint. The TG-induced permeability response was inhibited by 60% with 27 µM Gd3+, a response that was maintained up to 50 µM Gd3+ (data not shown). We were not able to test higher doses due to limitations in Gd3+ solubility. On the basis of these results, we chose 30 µM Gd3+ for the subsequent studies. Pretreatment of the rat lung with 30 µM Gd3+ did not prevent 14,15-EET-induced increase in Kf,c but blocked TG-induced lung injury. As we could not achieve 100% inhibition of the permeability response induced by 14,15-EET with 30 µM Gd3+, we cannot conclude from this experiment that the permeability response to the eicosanoid in rat lung is due to Ca2+ entry. We sought a more definitive answer by using a low-Ca2+ buffer. Chetham et al. (4) previously reported that a low-Ca2+ buffer prevented TG-induced EC injury in rat lung. In our hands, a dose-response curve revealed that 20 µM was the lowest [Ca2+] that allowed stability of the lung, as evidenced by stable Kf,c during a 2-h period. As expected, the permeability response to TG was blocked in the presence of a low-Ca2+ buffer. Importantly, we showed that readdition of Ca2+ to the perfusate to achieve physiological levels (2.2 mM) resulted in TG-induced lung injury in the same lungs. Similarly to TG, 14,15-EET increased EC permeability following readdition of Ca2+, supporting the notion that EETs promote Ca2+ entry in rat lung EC and that this contributes to the EET-induced increase in permeability. However, there was a tendency for Kf,c to increase following EET administration, even in the low [Ca2+] perfusate, a response not significantly different from that seen after Ca2+ add-back. These data suggest that 14,15-EET also impacts a Ca2+-independent pathway involved in the regulation of EC permeability.

The observation that the EET-induced increase in EC permeability is dependent on both Ca2+ entry and other Ca2+-independent mechanisms does not allow us to confirm the molecular target of these eicosanoids. For example, EETs could act via direct receptor-mediated activation of Ca2+ channels or activate a signaling cascade upstream of the Ca2+ entry pathway. EETs have been shown to evoke hyperpolarization in EC via activation of Ca2+-dependent potassium channels, an effect that would enhance the electrochemical gradient for Ca2+ entry (16). Although we have shown that potassium channel activation did not play a role in the canine lung (18), it remains possible that this is a critical step in rat lung EC. Potential targets that might plausibly be involved in the Ca2+-independent regulation of EC permeability by EETs include tyrosine kinases and the extracellular regulated protein kinases (ERK). Several reports provide evidence that EETs activate tyrosine kinases and thus downstream kinases such as MAPK and ERK in pig aortic and coronary artery EC (8, 15, 27). These observations are relevant, since ERK activation has been linked to Ca2+-dependent and -independent increases in pulmonary EC permeability (10, 45). Identification of the specific pathways involved in mediating EETs' effect on EC permeability will require additional experiments beyond the scope of the present study.

Finally, EETs have been postulated to act as soluble factors involved in capacitative Ca2+ entry (1, 13). In bovine coronary artery and aortic EC, P-450 inhibitors blocked TG-induced Ca2+ entry, and 5,6-EET mimicked the effect of TG on Ca2+ entry (12, 13). In canine lung, P-450 inhibitors prevented acute lung injury following challenge with TG and limited edema formation following treatment with ethchlorvynol, suggesting that under some conditions, endogenous EET synthesis does participate in regulation of lung EC permeability (18, 38). On the basis of these studies and our current observation that the effect of 14,15-EET on EC permeability was in part dependent on Ca2+ entry, the hypothesis that EETs are soluble factors involved in capacitative Ca2+ entry in the rat lung was rational. However, these P-450 metabolites do not appear to mediate changes in EC permeability subsequent to store depletion in rat lung. Neither the nonspecific PLA2 inhibitor mepacrine or the more selective inhibitor MAFP was able to attenuate the TG-induced increase in EC permeability, suggesting that AA availability was not a factor in this process. Similarly, if metabolism of AA via P-450 epoxygenases was involved in regulating Ca2+ entry after TG, then 17-ODYA and PPOH should have blocked the TG-induced permeability response (24, 32). Again, we found no blunting of the TG-induced permeability response in lungs pretreated with these inhibitors. Thus in contrast to our previous observation in canine lung (18), there appears to be no role for P-450 epoxygenases in the response to TG in the rat lung. Although mepacrine enhanced the TG-induced permeability response, we believe that this is likely attributed to the relative nonspecificity of this inhibitor (6). Confirmation of our conclusion that EETs do not mediate the permeability response to TG was obtained by subsequent experiments that showed that inhibition of EET metabolism, achieved by concomitant treatment with the cyclooxygenase inhibitor ibuprofen and the soluble epoxide hydrolase inhibitor DCU, did not enhance the TG-induced increase in Kf,c. Thus despite the fact that 5,6- and 14,15-EET increase EC permeability, and 14,15-EET does so in a partially Ca2+ entry-dependent fashion in the rat lung, the mechanism linking store depletion and capacitative Ca2+ entry in rat pulmonary EC does not involve EETs.

In summary, we have demonstrated that EETs increase EC permeability in rat lung in a regiospecific and Ca2+ entry-dependent manner, although a Ca2+-independent pathway appears to be activated in parallel. On the basis of the observation that the response to TG does not involve EETs, we speculate that the increase in permeability evoked by EETs is independent of store depletion. Further work will be needed to establish a clear view of how these epoxygenase metabolites participate in regulation of endothelial barrier function. Specifically, the signaling mechanism(s) underlying the Ca2+-independent permeability response and the molecular identity of the Ca2+ entry modulated by EETs remain to be elucidated.


    ACKNOWLEDGMENTS
 
GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-61955, a grant from the Norwegian Research Council, and American Heart Association Southeast Affiliate predoctoral fellowship 0315049B.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. I. Townsley, Dept. of Physiology, MSB 3074, Univ. of So. Alabama, Mobile, AL 36688 (E-mail: mtownsley{at}usouthal.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.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Alvarez J, Montero M, and Garcia-Sancho J. Cytochrome P450 may link intracellular Ca++ stores with plasma membrane Ca++ influx. Biochem J 274: 193-197, 1991.[ISI][Medline]
  2. Brough GH, Wu S, Cioffi D, Moore TM, Li M, Dean N, and Stevens T. Contribution of endogenously expressed Trp1 to a Ca++-selective, store-operated Ca++ entry pathway. FASEB J 15: 1727-1738, 2001.[Abstract/Free Full Text]
  3. Caldwell RA, Clemo HF, and Baumgarten CM. Using gadolinium to identify stretch-activated channels: technical considerations. Am J Physiol Cell Physiol 275: C619-C621, 1998.[Abstract/Free Full Text]
  4. Chetham PM, Babal P, Bridges JP, Moore TM, and Stevens T. Segmental regulation of pulmonary vascular permeability by store-operated Ca2+ entry. Am J Physiol Lung Cell Mol Physiol 276: L41-L50, 1999.[Abstract/Free Full Text]
  5. Drake R, Gaar KA, and Taylor AE. Estimation of the filtration coefficient of pulmonary exchange vessels. Am J Physiol Heart Circ Physiol 234: H266-H274, 1978.[Abstract/Free Full Text]
  6. Dulin NO, Alexander LD, Harwalkar S, Falck JR, and Douglas JG. Phospholipase A2-mediated activation of mitogen-activated protein kinase by angiotensin II. Proc Natl Acad Sci USA 95: 8098-8102, 1998.[Abstract/Free Full Text]
  7. Fang X, Kaduce TL, Weintraub NL, Harmon S, Teesch LM, Morisseau C, Thompson DA, Hammock BD, and Spector AA. Pathways of epoxyeicosatrienoic acid metabolism in endothelial cells. Implications for the vascular effects of soluble epoxide hydrolase inhibition. J Biol Chem 276: 14867-14874, 2001.[Abstract/Free Full Text]
  8. Fleming I, Fisslthaler B, Michaelis UR, Kiss L, Popp R, and Busse R. The coronary endothelium-derived hyperpolarizing factor (EDHF) stimulates multiple signalling pathways and proliferation in vascular cells. Pflügers Arch 442: 511-518, 2001.[CrossRef][ISI][Medline]
  9. Garcia JG, Schaphorst KL, Shi S, Verin AD, Hart CM, Callahan KS, and Patterson CE. Mechanisms of ionomycin-induced endothelial cell barrier dysfunction. Am J Physiol Lung Cell Mol Physiol 273: L172-L184, 1997.[Abstract/Free Full Text]
  10. Garcia JG, Schaphorst KL, Verin AD, Vepa S, Patterson CE, and Natarajan V. Diperoxovanadate alters endothelial cell focal contacts and barrier function: role of tyrosine phosphorylation. J Appl Physiol 89: 2333-2343, 2000.[Abstract/Free Full Text]
  11. Gjerde EB, Stevens T, and Townsley MI. 14,15-Epoxyeicosatrienoic acid activates store-operated calcium channels in rat pulmonary microvascular endothelial cells (Abstract). FASEB J late-breaking abstracts: 8, 2003.
  12. Graber MN, Alfonso A, and Gill DL. Recovery of Ca++ pools and growth in Ca++ pool-depleted cells is mediated by specific epoxyeicosatrienoic acids derived from arachidonic acid. J Biol Chem 272: 29546-29553, 1997.[Abstract/Free Full Text]
  13. Graier WF, Simecek S, and Sturek M. Cytochrome P450 monooxygenase-regulated signalling of Ca++ entry in human and bovine endothelial cells. J Physiol 482: 259-274, 1995.[Abstract]
  14. Halaszovich CR, Zitt C, Jungling E, and Luckhoff A. Inhibition of Trp3 channels by lanthanides. Block from the cytosolic side of the plasma membrane. J Biol Chem 275: 37423-37428, 2000.[Abstract/Free Full Text]
  15. Hoebel BG and Graier WF. 11,12-Epoxyeicosatrienoic acid stimulates tyrosine kinase activity in porcine aortic endothelial cells. Eur J Pharmacol 346: 115-117, 1998.[CrossRef][ISI][Medline]
  16. Hoebel BG, Kostner GM, and Graier WF. Activation of microsomal cytochrome P450 mono-oxygenase by Ca2+ store depletion and its contribution to Ca2+ entry in porcine aortic endothelial cells. Br J Pharmacol 121: 1579-1588, 1997.[Abstract]
  17. Ivey CL, Roy BJ, and Townsley MI. Ablation of lung endothelial injury after pacing-induced heart failure is related to alterations in Ca2+ signaling. Am J Physiol Heart Circ Physiol 275: H844-H851, 1998.[Abstract/Free Full Text]
  18. Ivey CL, Stephenson AH, and Townsley MI. Involvement of cytochrome P-450 enzyme activity in the control of microvascular permeability in canine lung. Am J Physiol Lung Cell Mol Physiol 275: L756-L763, 1998.[Abstract/Free Full Text]
  19. Khimenko PL and Taylor AE. Segmental microvascular permeability in ischemia-reperfusion injury in rat lung. Am J Physiol Lung Cell Mol Physiol 276: L958-L960, 1999.[Abstract/Free Full Text]
  20. Kiss L, Schutte H, Mayer K, Grimm H, Padberg W, Seeger W, and Grimminger F. Synthesis of arachidonic acid-derived lipoxygenase and cytochrome P450 products in the intact human lung vasculature. Am J Respir Crit Care Med 161: 1917-1923, 2000.[Abstract/Free Full Text]
  21. Mombouli JV, Holzmann S, Kostner GM, and Graier WF. Potentiation of Ca++ signaling in endothelial cells by 11,12-epoxyeicosatrienoic acid. J Cardiovasc Pharmacol 33: 779-784, 1999.[CrossRef][ISI][Medline]
  22. Moore TM, Chetham PM, Kelly JJ, and Stevens T. Signal transduction and regulation of lung endothelial cell permeability. Interaction between calcium and cAMP. Am J Physiol Lung Cell Mol Physiol 275: L203-L222, 1998.[Abstract/Free Full Text]
  23. Moore TM, Khimenko P, Adkins WK, Miyasaka M, and Taylor AE. Adhesion molecules contribute to ischemia and reperfusion-induced injury in the isolated rat lung. J Appl Physiol 78: 2245-2252, 1995.[Abstract/Free Full Text]
  24. Murray M and Reidy GF. Selectivity in the inhibition of mammalian cytochromes P450 by chemical agents. Pharmacol Rev 42: 85-101, 1990.[ISI][Medline]
  25. Nilius B and Droogmans G. Ion channels and their functional role in vascular endothelium. Physiol Rev 81: 1415-1459, 2001.[Abstract/Free Full Text]
  26. Norwood N, Moore TM, Dean DA, Bhattacharjee R, Li M, and Stevens T. Store-operated calcium entry and increased endothelial cell permeability. Am J Physiol Lung Cell Mol Physiol 279: L815-L824, 2000.[Abstract/Free Full Text]
  27. Popp R, Brandes RP, Ott G, Busse R, and Fleming I. Dynamic modulation of interendothelial gap junctional communication by 11,12-epoxyeicosatrienoic acid. Circ Res 90: 800-806, 2002.[Abstract/Free Full Text]
  28. Putney JW Jr. A model for receptor-regulated calcium entry. Cell Calcium 7: 1-12, 1986.[ISI][Medline]
  29. Rippe B, Parker JC, Townsley MI, Mortillaro NA, and Taylor AE. Segmental vascular resistances and compliances in dog lung. J Appl Physiol 62: 1206-1215, 1987.[Abstract/Free Full Text]
  30. Rippe B, Townsley MI, and Taylor AE. Effects of plasma- and cell-free perfusate on filtration coefficient of perfused canine lungs. J Appl Physiol 58: 1521-1527, 1985.[Abstract/Free Full Text]
  31. Roberts MF. Phospholipases: structural and functional motifs for working at an interface. FASEB J 10: 1159-1172, 1996.[Abstract/Free Full Text]
  32. Roman RJ. P-450 metabolites of arachidonic acid in the control of cardiovascular function. Physiol Rev 82: 131-185, 2002.[Abstract/Free Full Text]
  33. Rzigalinski BA, Willoughby KA, Hoffman SW, Falck JR, and Ellis EF. Calcium influx factor, further evidence it is 5,6-epoxyeicosatrienoic acid. J Biol Chem 274: 175-182, 1999.[Abstract/Free Full Text]
  34. Sato S, Kato S, Arisaka Y, Takahashi H, Takahashi K, and Tomoike H. Changes in pulmonary hemodynamics during normoxia and hypoxia in awake rats treated with intratracheal bleomycin. Tohoku J Exp Med 169: 233-244, 1993.[ISI][Medline]
  35. Shibamoto T, Parker JC, Taylor AE, and Townsley MI. Derecruitment of filtration surface area in paraquat-injured isolated dog lungs. J Appl Physiol 68: 1581-1589, 1990.[Abstract/Free Full Text]
  36. Stephenson AH, Sprague RS, and Lonigro AJ. 5,6-Epoxyeicosatrienoic acid reduces increases in pulmonary vascular resistance in the dog. Am J Physiol Heart Circ Physiol 275: H100-H109, 1998.[Abstract/Free Full Text]
  37. Stephenson AH, Sprague RS, Losapio JL, and Lonigro AJ. Differential effects of 5,6-EET on segmental pulmonary vasoactivity in the rabbit. Am J Physiol Heart Circ Physiol 284: H2153-H2161, 2003.[Abstract/Free Full Text]
  38. Stephenson AH, Sprague RS, Weintraub NL, McMurdo L, and Lonigro AJ. Inhibition of cytochrome P-450 attenuates hypoxemia of acute lung injury in dogs. Am J Physiol Heart Circ Physiol 270: H1355-H1362, 1996.[Abstract/Free Full Text]
  39. Stevens T, Garcia JG, Shasby DM, Bhattacharya J, and Malik AB. Mechanisms regulating endothelial cell barrier function. Am J Physiol Lung Cell Mol Physiol 279: L419-L422, 2000.[Abstract/Free Full Text]
  40. Thastrup O, Cullen P, Drobak B, Hanley M, Dawson A, and Durbak B. Thapsigargin, a tumor promoter, discharges intracellular calcium stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc Natl Acad Sci USA 87: 2466-2470, 1990.[Abstract]
  41. Townsley MI, Korthuis RJ, Rippe B, Parker JC, and Taylor AE. Validation of double vascular occlusion method for Pc,i in lung and skeletal muscle. J Appl Physiol 61: 127-132, 1986.[Abstract/Free Full Text]
  42. Townsley MI, Parker JC, Korthuis RJ, and Taylor AE. Alterations in hemodynamics and Kf,c during lung mass resection. J Appl Physiol 63: 2460-2466, 1987.[Abstract/Free Full Text]
  43. Townsley MI, Pitts VH, Ardell JL, Zhao Z, and Johnson WH Jr. Altered pulmonary microvascular reactivity to norepinephrine in canine pacing-induced heart failure. Circ Res 75: 347-356, 1994.[Abstract]
  44. Trebak M, Bird GS, McKay RR, and Putney JW Jr. Comparison of human TRPC3 channels in receptor-activated and store-operated modes. Differential sensitivity to channel blockers suggests fundamental differences in channel composition. J Biol Chem 277: 21617-21623, 2002.[Abstract/Free Full Text]
  45. Verin AD, Liu F, Bogatcheva N, Borbiev T, Hershenson MB, Wang P, and Garcia JG. Role of ras-dependent ERK activation in phorbol ester-induced endothelial cell barrier dysfunction. Am J Physiol Lung Cell Mol Physiol 279: L360-L370, 2000.[Abstract/Free Full Text]
  46. Wang MH, Brand-Schieber E, Zand BA, Nguyen X, Falck JR, Balu N, and Schwartzman ML. Cytochrome P450-derived arachidonic acid metabolism in the rat kidney: characterization of selective inhibitors. J Pharmacol Exp Ther 284: 966-973, 1998.[Abstract/Free Full Text]
  47. Wu S, Sangerman J, Li M, Brough GH, Goodman SR, and Stevens T. Essential control of an endothelial cell ISOC by the spectrin membrane skeleton. J Cell Biol 154: 1225-1233, 2001.[Abstract/Free Full Text]
  48. Xiao YF, Huang L, and Morgan JP. Cytochrome P450: a novel system modulating Ca++ channels and contraction in mammalian heart cells. J Physiol 508: 777-792, 1998.[Abstract/Free Full Text]
  49. Yaghi A, Webb CD, Scott JA, Mehta S, Bend JR, and McCormack DG. Cytochrome P450 metabolites of arachidonic acid but not cyclooxygenase-2 metabolites contribute to the pulmonary vascular hyporeactivity in rats with acute Pseudomonas pneumonia. J Pharmacol Exp Ther 297: 479-488, 2001.[Abstract/Free Full Text]
  50. Zeldin DC. Epoxygenase pathways of arachidonic acid metabolism. J Biol Chem 276: 36059-36062, 2001.[Free Full Text]
  51. Zhu D, Bousamra M, Zeldin DC, Falck JR, Townsley M, Harder DR, Roman RJ, and Jacobs ER. Epoxyeicosatrienoic acids constrict isolated pressurized rabbit pulmonary arteries. Am J Physiol Lung Cell Mol Physiol 278: L335-L343, 2000.[Abstract/Free Full Text]