Involvement of cytochrome P-450 enzyme activity in the control of microvascular permeability in canine lung

Claire L. Ivey1, Alan H. Stephenson2, and Mary I. Townsley1

1 Department of Physiology, University of South Alabama, Mobile, Alabama 36688; and 2 Department of Pharmacological and Physiological Science, St. Louis University School of Medicine, St. Louis, Missouri 63104

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Products of cytochrome P-450 enzymes may play a role in capacitative Ca2+ entry in endothelial cells, which can promote a rise in vascular permeability. Thapsigargin (150 nM) stimulated capacitative Ca2+ entry and increased the capillary filtration coefficient (Kf,c) in isolated normal canine lung lobes. Pretreatment of the lobes with cytochrome P-450 inhibitors clotrimazole (10 µM) or 17-octadecynoic acid (5 µM) abolished the thapsigargin-induced increases in Kf,c. Because clotrimazole also blocks Ca2+-activated K+ channels, the K+-channel blocker tetraethylammonium (10 mM) was used to ensure that permeability was not influenced by this mechanism. Tetraethylammonium did not affect thapsigargin-induced permeability. The effects of the cytochrome P-450 arachidonic acid metabolite 5,6-epoxyeicosatrienoic acid (EET) were also investigated in lobes taken from control dogs and dogs with pacing-induced heart failure (paced at 245 beats/min for 4 wk). 5,6-EET (10 µM) significantly increased Kf,c in lobes from the control but not from the paced animals. We conclude that cytochrome P-450 metabolites are involved in mediating microvascular permeability in normal canine lungs, but an absence of 5,6-EET after heart failure does not explain the resistance of lungs from these animals to permeability changes.

capacitative calcium entry; epoxyeicosatrienoic acid; pulmonary endothelium; congestive heart failure; capillary filtration coefficient

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE MICROVASCULAR ENDOTHELIUM forms the primary barrier that regulates movement of fluid and macromolecules from blood to tissues. Preservation of this barrier is essential such that its disruption leads to intracellular gap formation and increased microvascular permeability, resulting in increased fluid transfer into the interstitium and edema formation. It is well documented that agents that raise endothelial cell Ca2+ concentrations also increase microvascular permeability via Ca2+-induced stimulation of endothelial cell contraction (14, 15, 27, 40) and inhibition of cell-matrix and cell-cell tethering (15, 28). One mechanism by which cytosolic Ca2+ concentration ([Ca2+]i) can be increased is via a capacitative Ca2+ entry (CCE) pathway (32, 33). CCE can be promoted via G protein-mediated activation of phospholipase (PL) C, resulting in the generation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] (4), such as after challenge with angiotensin II (ANG II) (25, 49). Binding of Ins(1,4,5)P3 to a receptor on intracellular Ca2+ stores initiates release of Ca2+ into the cytosol, which, in turn, appears to be the stimulus for further Ca2+ to enter the cell via a plasma membrane store-operated Ca2+ channel (SOC) (32, 33). The plant alkaloid thapsigargin can also be used to evoke CCE by inhibiting Ca2+ uptake into intracellular stores (43). Previously, Ivey et al. (21) showed that thapsigargin increases pulmonary microvascular permeability in normal canine lung lobes but fails to modify permeability in lung lobes taken from dogs after pacing-induced heart failure. Similar observations are seen with ANG II (37). Therefore, it appears that after heart failure, adaptations occur in the lungs such that they become resistant to changes in permeability induced by mediators stimulating CCE.

The precise mechanism linking intracellular store depletion with CCE is still unclear. However, it may involve the release of a cytosolic factor that diffuses to the plasma membrane to open the SOC (9, 29, 34). It has been proposed that arachidonic acid (AA) metabolites formed via cytochrome P-450 monoxygenase enzymes are important in the signaling pathway to open SOCs (2, 17). Free AA can be metabolized by cytochrome P-450 via epoxidation to form epoxyeicosatrienoic acids (EETs) or via omega  and omega -1 oxidation to form 19- and 20-hydroxyeicosatetraenoic acids (HETEs), respectively (18). Graier et al. (17) recently proposed that generation of the epoxygenase metabolite 5,6-EET is a key step in activating CCE after the depletion of endothelial Ca2+ stores.

Because the cytochrome P-450 metabolite 5,6-EET is known to be synthesized in normal canine lungs (42), we carried out experiments to investigate a possible role for 5,6-EET in mediating canine pulmonary microvascular permeability. The effects of cytochrome P-450 inhibition on thapsigargin-induced changes in vascular permeability were monitored in control canine lungs. This was done with two cytochrome P-450 inhibitors, clotrimazole and 17-octadecynoic acid (17-ODYA). At the concentrations used, clotrimazole inhibits the epoxygenase pathway of cytochrome P-450 AA metabolism, whereas 17-ODYA inhibits both the epoxygenase and omega -hydroxylase pathways (54). Clotrimazole also has the ability to block Ca2+-activated K+ channels (3, 6), which can result in plasma membrane depolarization, thereby inhibiting Ca2+ influx into cells. Therefore, additional experiments were performed with the nonspecific K+-channel blocker tetraethylammonium (TEA) to ensure that permeability was not affected by this mechanism. As a further test of cytochrome P-450 involvement in mediating pulmonary permeability and to investigate whether this may be altered after pacing-induced heart failure, the effects of 5,6-EET were studied in isolated lung lobes taken from both control and paced dogs. Our data suggest that cytochrome P-450 metabolites are involved in pathways mediating pulmonary microvascular permeability in normal canine lungs. The resistance of heart failure lungs to injury does not, however, appear to be due to lack of production of 5,6-EET.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Pacing Model of Heart Failure

Conditioned, microfilaria-negative mongrel dogs (n = 5) were anesthetized with pentobarbital sodium (30 mg/kg iv) to allow insertion of a transvenous pacing lead (unipolar; model 4011, Medtronic) into the right ventricle via the right jugular vein. Correct placement of the pacing lead was confirmed by the ability to capture ventricular rate. A programmable generator (model 8329, Medtronic) was inserted into a subcutaneous pocket anterior to the first rib and attached to the pacing lead. All surgery was done under sterile conditions. Antibiotics (500 mg of cefazolin twice daily) were administered orally for 5 days postoperatively. Wound healing was checked daily. The animals were allowed to eat and drink ad libitum after surgery and during the pacing period.

A baseline echocardiogram was performed 1-2 days after surgery, after which pacing was initiated at a ventricular rate of 245 beats/min (21, 37, 45, 48). Maintenance of pacing was checked daily by auscultation. Echocardiograms were done at regular intervals to assess cardiac function. Pacing was continued for 29.2 ± 1.2 days during which time the left ventricular shortening fraction, measured in sinus rhythm, was significantly reduced from baseline.

Terminal Experiment

Conditioned, microfilaria-negative mongrel dogs were used as controls (n = 27). Both paced and control dogs were fasted overnight before the terminal experiments. An intravenous line was inserted, and anesthesia was induced with pentobarbital sodium (<15 mg/kg iv in paced dogs; 30 mg/kg iv in control dogs). Previous studies (45, 48) have shown that this regimen produces a surgical plane of anesthesia in paced dogs without accompanying cardiovascular collapse. The dogs were orally intubated and placed on a Harvard animal respirator at a rate of 15 breaths/min and a tidal volume of 15 ml/kg. alpha -Chloralose was administered as necessary to maintain a surgical plane of anesthesia. A carotid arterial line was inserted to measure systemic arterial blood pressure (paced dogs only) and to withdraw blood. In paced dogs, a catheter was placed in the jugular vein and advanced to the pulmonary artery for measurement of pulmonary arterial pressure (Pa). Placement of the lines was confirmed by waveforms.

Isolated Lung Preparation

The left chest was opened at the fifth intercostal space. The left upper and middle lobes were excised for measurement of blood-free extravascular lung water (EVLW). Loose ties were placed around the left main pulmonary artery and left bronchus. All dogs were heparinized with 10,000 units of heparin intravenously. Ten minutes postheparinization, the lower left lung lobe was removed. The lobar artery, vein, and bronchus were cannulated with plastic cannulas. The lobe was then suspended from a counterbalanced force transducer (Grass FT-10), and the system was calibrated with a standard weight. The lobes were perfused with 200 ml of autologous blood mixed with 100 ml of balanced Earle's buffer and ventilated with 30% O2-5% CO2 with a Harvard animal respirator at a rate of 6-8 breaths/min, a peak inspiratory pressure of 8-10 cmH2O, and an end-expiratory pressure of 2-3 cmH2O. Blood temperature was maintained at 37°C. Blood gases were measured, and the pH was corrected to 7.35-7.40 as necessary with the addition of sodium bicarbonate.

Lobar Hemodynamics

Thin catheters were placed in the lobar arterial and venous lines to measure Pa and venous pressure (Pv), respectively. The zero point was set at the lung hilum. Pressures and lobe weight were continuously recorded with a Grass model 7 polygraph. Pv was set to 4-5 cmH2O by adjusting the height of the venous reservoir. Blood flow (Q) was increased to the maximal value that would keep the lobe in an isogravimetric state (neither losing nor gaining weight) and remained at that level throughout the remainder of the experiment. Capillary pressure (Pc) was measured by the double vascular occlusion technique (46). At the end of the experiment, Q was measured by a timed collection of blood into a graduated cylinder. Total pulmonary resistance (RT) was calculated as (Pa - Pv)/Q. Precapillary (arterial; Ra) and postcapillary (venous; Rv) resistances were determined as (Pa - Pc)/Q and (Pc - Pv)/Q, respectively. Total vascular compliance (CT) was determined via the rapid venous occlusion technique (22) and calculated as CT = Q/(the linear rate of Delta Pv/Delta t after rapid venous occlusion), where t is time.

Evaluation of Permeability and Transvascular Fluid Exchange

The capillary filtration coefficient (Kf,c) was determined as a measure of microvascular permeability at the beginning and end of each experiment. Pv was increased by 8-10 cmH2O for a period of 15 min, causing the lobe to gain weight. The rate of weight gain (Delta W/Delta t) becomes constant after ~10 min. The Delta W/Delta t was calculated for minutes 13-15, and the result was used in the following equation to calculate Kf,c as (Delta W/Delta t)/Delta Pc. Pc was determined by the difference in Pc before and at the end of the 15-min increased Pv period. Kf,c is expressed as milliliters per minute per centimeter of H2O per 100 g of wet weight. At the end of the Kf,c measurement, Pv was returned to the baseline level.

EVLW was measured as follows (30, 51). The lobes were homogenized with 100 ml of distilled water, then samples of the homogenate were centrifuged for 1 h at 27,200 g to obtain lung supernatant. Samples of blood, homogenate, and supernatant were weighed and then dried to a constant weight. Lung water was corrected for blood water with these weights and measurements of total hemoglobin (5) in the blood and supernatant. EVLW is reported as milliliters per gram of blood-free dry weight.

Synthesis of 5,6-EET

Selective epoxidation of AA to 5,6-EET was achieved with the method of Corey et al. (8). Briefly, AA (10 mg) was incubated with potassium triiodide (8 eq) and potassium bicarbonate (5 eq) in tetrahydrofuran-water (1.5:1) under N2 gas for 16 h at 4°C. Excess iodine was removed by dropwise addition of saturated sodium sulfite (500 µl) until the solution cleared. The combined organic extracts were dried by vacuum centrifugation (Savant), dissolved in 1 ml of tetrahydrofuran, and incubated with 500 µl of lithium hydroxide (0.2 M) with constant stirring for 3 h at 25°C. The reaction mixture containing the 5,6-EET formed was acidified with formic acid (pH 4.0), extracted three times with ethyl acetate (2 ml), washed once with H2O (1 ml), and purified by reverse-phase HPLC with a Nucleosil C18 column (5 µm, 4.6 × 250 mm) with a linear gradient from 50% water in acetonitrile-acetic acid (999:1) to 100% acetonitrile-acetic acid (999:1) over 40 min at 1 ml/min. Eluate containing 5,6-EET was collected, evaporated to dryness, and stored under N2 gas in hexane at -80°C. The identity of the 5,6-EET synthesized was confirmed by comparison with an authentic standard (Cayman Chemical) with HPLC and gas chromatography-mass spectrometry as previously reported (42). In its free-acid form, 5,6-EET readily decomposes to 5,6-dihydroxyeicosatrienoic acid and the corresponding delta -lactone. Therefore, before use, 5,6-EET was repurified by reverse-phase HPLC.

Isolated Lung Protocols

Initial pressures, CT, and Kf,c were determined for all lobes. The experimental protocols, described below, were begun after the lobes had stabilized after these baseline measurements were made. At the end of the experiment, final EVLW was measured for each perfused lobe as described in Evaluation of Permeability and Transvascular Fluid Exchange.

Thapsigargin. The effect of thapsigargin on microvascular permeability was assessed in six control lobes. In these experiments, ibuprofen (10 µg/ml perfusate) was added to the venous reservoir to attenuate thapsigargin-induced vasoconstriction. After 30 min, thapsigargin was added to the perfusate to give a final concentration of 150 nM (21). Final pressure, CT, and Kf,c measurements were made after 1 h. In lobes that had a prolonged pressor response to thapsigargin (i.e., >1 h), final measurements were made when Pa stabilized. Vascular resistances were measured before each drug addition. All experiments involving thapsigargin were performed in the dark due to its sensitivity to light.

Cytochrome P-450 inhibition and thapsigargin. The effect of cytochrome P-450 inhibition on the thapsigargin-induced permeability change was assessed in control lobes. In addition to ibuprofen (10 µg/ml), one of two different cytochrome P-450 inhibitors, either clotrimazole (n = 5 lobes) or 17-ODYA (n = 6 lobes), was added to the venous reservoir to give final concentrations of 10 and 5 µM, respectively. After 30 min, thapsigargin was added to the perfusate at a final concentration of 150 nM. Final pressure, CT, and Kf,c measurements were made after 1 h or when pressures were stable. Vascular resistances were measured before each drug addition.

TEA and thapsigargin. The effects of Ca2+-activated K+-channel blockade on thapsigargin-induced permeability were investigated in five control lobes with TEA. TEA was added to the venous reservoir (final concentration 10 mM) with ibuprofen (10 µg/ml perfusate). After 30 min, thapsigargin was added to the perfusate at a final concentration of 150 nM. Final pressure, CT, and Kf,c measurements were made after 1 h or when pressures were stable (see above). Vascular resistances were measured before each drug addition.

5,6-EET. The effects of 5,6-EET on microvascular permeability were assessed in lung lobes taken from five control and five paced dogs. 5,6-EET was added to the perfusate, resulting in a final concentration of 10 µM (due to the rapid degradation of 5,6-EET, this concentration represents the initial concentration in the perfusate). Measurements of pressure, CT, and Kf,c were made at 1 and 2 h after 5,6-EET administration. These experiments were performed in the dark to allow for possible sensitivity of 5,6-EET to light.

Drugs

Clotrimazole, ibuprofen, TEA, and 17-ODYA were obtained from Sigma. Clotrimazole, ibuprofen, and 17-ODYA were dissolved in 95% ethanol and kept at room temperature. Fresh solutions of TEA were made in 0.9% sodium chloride on each day of experimentation. Thapsigargin was purchased from RBI and dissolved in DMSO, and aliquots were kept at -20°C. Aliquots of 5,6-EET were stored at -80°C and dissolved in absolute ethanol immediately before use.

Statistics

Data are presented as means ± SE. Significant differences were evaluated by ANOVA or, when appropriate, by Student's t-test to identify specific differences within and between groups.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals in the heart failure group were paced for an average of 29.2 ± 1.2 days, resulting in a decrease in left ventricular shortening fraction from 32.8 ± 0.9 to 20.0 ± 1.3% (P < 0.05). Body weight in this group before pacing (21.6 ± 0.8 kg) was not different from that at the time of the terminal experiment (21.4 ± 0.6 kg). Measurements of cardiovascular function in the paced group after anesthesia showed that the in vivo systemic Pa was 109 ± 7.8 mmHg (n = 4 dogs), the central Pv was 6.5 ± 3.5 mmHg (n = 4 dogs), the pulmonary Pa was 35.5 ± 6.9 mmHg (n = 4 dogs), and the pulmonary wedge pressure was 24.7 ± 4.9 mmHg (n = 4 dogs). These results are comparable with those previously reported in paced dogs (37, 48). Although in vivo measurements were not made in the current control dogs, the initial EVLW (see below) was no different from that of previous control dogs with normal pulmonary vascular pressures (48). There was no difference between the body weight of control (21.7 ± 0.5 kg) and paced (21.4 ± 0.6 kg) dogs at the time of the terminal experiment. The lungs of the paced animals had a higher initial EVLW (7.4 ± 0.7 ml/g) than the control group (4.2 ± 0.1 ml/g; P < 0.05). Table 1 shows the baseline data for all isolated lung lobes. Roy et al. (37) and Townsley et al. (48) previously showed that baseline resistances in the paced lobes were significantly higher than those in the control lobes, and the data in this study confirm that.

                              
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Table 1.   Baseline hemodynamic measurements in isolated lung lobes

Table 2 shows that administration of thapsigargin to control lung lobes had no effect on Ra but caused a marked venoconstriction, with a significant increase in Rv (P < 0.05). Pretreatment of the lobes with either one of the two cytochrome P-450 inhibitors (clotrimazole or 17-ODYA) or the K+-channel blocker (TEA) did not affect baseline Ra or Rv (data not shown) nor did it influence thapsigargin-induced changes in these parameters (Table 2). As previously described (21, 37, 45, 48), baseline Ra and Rv were both augmented in the paced lobes compared with the control lobes (Fig. 1). Administration of 5,6-EET had no effect on Ra or Rv in either the control or paced group (P > 0.05).

                              
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Table 2.   Vascular resistance measurements from isolated control lung lobes before and after different drug treatments


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Fig. 1.   Effects of epoxygenase metabolite 5,6-epoxyeicosatrienoic acid (EET) on arterial (Ra; A) and venous (Rv; B) resistances 1 and 2 h after administration in lung lobes removed from control (n = 5; open bars) and paced (n = 5; solid bars) dogs. ** Significant difference from control values at the same time point, P < 0.05.

Figure 2 demonstrates that thapsigargin promoted a prominent rise in Kf,c in control lobes as previously reported (21). This response was measured 100.8 ± 10.2 min after thapsigargin administration. Inhibition of cytochrome P-450 enzymes by addition of the imidazole antimycotic clotrimazole or the suicide-substrate inhibitor 17-ODYA significantly attenuated thapsigargin-induced changes in Kf,c (Fig. 2) after 84.8 ± 9.5 and 107.1 ± 5.8 min, respectively. There was no difference in the mean time between thapsigargin administration and the final Kf,c measurements in the above three groups (P > 0.05).


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Fig. 2.   Effects of cytochrome P-450 inhibitors clotrimazole and 17-octadecynoic acid (17-ODYA) on thapsigargin (TG)-induced changes in capillary filtration coefficient (Kf,c) in lung lobes from control dogs. TG (150 nM) was administered alone (n = 6 lobes; open bars) or 30 min after pretreatment with either clotrimazole (10 µM; n = 5 lobes; hatched bars) or 17-ODYA (5 µM; n = 6 lobes; solid bars). Significant difference (P < 0.05) from: ** baseline within the same group; # no pretreatment at the same time point.

To ensure that the ability of clotrimazole to block Ca2+-activated K+ channels (3, 6) did not influence pulmonary endothelial permeability, control lobes were treated with the nonspecific K+-channel blocker TEA before thapsigargin administration. Figure 3 shows that pretreatment of control lobes with TEA had no effect on thapsigargin-induced permeability increases after 106.2 ± 7.1 min.


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Fig. 3.   Effects of K+-channel blocker tetraethylammonium (TEA) on TG-induced changes in Kf,c in lung lobes from control dogs. TG (150 nM) was administered alone (n = 6 lobes; open bars) or 30 min after pretreatment with TEA (10 mM; n = 5 lobes; solid bars). ** Significant difference from baseline within the same group.

There was no difference in baseline Kf,c between control and paced lobes (Fig. 4), supporting previous data (21, 37, 45, 48). However, 1 h after the administration of 5,6-EET in the control lobes, there was a significant rise in Kf,c (P < 0.05) that was still prominent after 2 h. In stark contrast, in the paced lobes, Kf,c was significantly attenuated after 5,6-EET at both time points (P < 0.05).


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Fig. 4.   Effects of epoxygenase metabolite 5,6-EET on Kf,c measured 1 and 2 h after administration in lung lobes removed from control (n = 5; open bars) and paced (n = 5; solid bars) dogs. Baseline Kf,c was not different between control and paced lobes. Significant difference from: *,** baseline within the same group; ** paced at the same time point.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The results from the present study support the notion that AA metabolites produced by cytochrome P-450 enzymes play a role in mediating increases in microvascular permeability in normal canine lungs. Evidence for this comes from the findings that 1) two mechanistically dissimilar cytochrome P-450 inhibitors prevented increases in permeability induced by thapsigargin and 2) administration of a product of the cytochrome P-450 monoxygenase metabolism of AA, 5,6-EET, promotes a rise in permeability. Supporting this notion, 5,6-EET, in addition to the other epoxygenase P-450 metabolites, is known to be produced by normal canine lungs (42).

Several studies have suggested that products of cytochrome P-450 AA metabolism provide the link between intracellular Ca2+ store depletion and the opening of plasma membrane SOCs in thymocytes (1), neutrophils (26), and, most relevant to this study, endothelial cells (17). CCE can be stimulated by G protein activation of PLC, leading to the generation of Ins(1,4,5)P3, which, in turn, binds to the intracellular Ca2+ stores (4). Release of the stored Ca2+ promotes the opening of the SOC via an unknown mechanism, allowing a further, more sustained increase in [Ca2+]i (32, 33). Activation of CCE has been demonstrated in vascular endothelium (10, 39, 50), in which large increases in [Ca2+]i can induce a rise in vascular permeability (14, 15, 27). In both human and bovine vascular endothelial cells, it has been shown that cytochrome P-450 inhibitors attenuate the sustained [Ca2+]i plateau response stimulated by store depletion with bradykinin, histamine, and thapsigargin (17).

Thapsigarin inhibits Ca2+-ATPase on intracellular Ca2+ stores, thus inducing CCE and increasing [Ca2+]i levels (43). Ivey et al. (21) previously showed that the addition of thapsigargin to the perfusate of control lung lobes markedly increases Kf,c. The data in this study support that finding. Because thapsigargin-induced increases in platelet and endothelial cell [Ca2+]i likely stimulate thromboxane synthesis via PLA2 activation, lobes were pretreated with ibuprofen before thapsigargin to attenuate the related pressor response. Note that thapsigargin still resulted in venoconstriction (Table 2) despite ibuprofen pretreatment, likely due to a direct effect of thapsigargin on venular smooth muscle (16). Nonetheless, the thapsigargin-induced rise in permeability is not thought to be due to any corresponding venoconstriction because the change in Pc is far below that already reported to be required to induce changes in Kf,c in this model (45). Furthermore, ibuprofen alone does not change permeability nor does it modify thapsigargin-mediated increases in permeability (data not shown). To investigate the role of cytochrome P-450 enzymes in mediating changes in endothelial barrier function, experiments were performed in which control lobes were pretreated with cytochrome P-450 inhibitors before thapsigargin. Two dissimilar inhibitors were used: clotrimazole and 17-ODYA. Clotrimazole is an imidazole antimycotic and, at the dose used in this study (10 µM), blocks only the cytochrome P-450 epoxygenase pathway for metabolism of AA, thus preventing EET formation. 17-ODYA is a chemically distinct compound that acts as a suicide-substrate inhibitor of cytochrome P-450 fatty acid metabolism. At the dose used in this study (5 µM), 17-ODYA inhibits the formation of both epoxygenase and 4-hydroxylase metabolites (54, 55). If the pathway for CCE relies on metabolites of cytochrome P-450 enzymes and thapsigargin increases pulmonary endothelial permeability via CCE, it follows that inhibition of cytochrome P-450 metabolism should block thapsigargin-induced increases in permeability. Our results show that cytochrome P-450 inhibition with either inhibitor does indeed abolish thapsigargin-induced rises in Kf,c.

The fact that both cytochrome P-450 inhibitors, clotrimazole and 17-ODYA, prevented increases in Kf,c is good evidence that cytochrome P-450 enzymes are required for permeability changes to occur when stimulating CCE via store depletion. Because clotrimazole selectively blocks EET production, these would appear to be the metabolites of importance. However, one major concern when using clotrimazole is its additional property of blocking Ca2+-activated K+ channels (3, 6, 38). This action can result in plasma membrane depolarization, which would reduce the driving gradient for Ca2+ entry, thus impeding increases in endothelial permeability. Further experiments were therefore carried out to ensure that this was not the mechanism by which clotrimazole attenuates microvascular permeability. Pretreatment of control lobes with the nonspecific K+-channel blocker TEA, at a dose of 10 mM, did not affect thapsigargin-induced increases in Kf,c, confirming that inhibition of thapsigargin-induced increases in permeability by clotrimazole is a result of cytochrome P-450 inhibition. Supporting this is the knowledge that 17-ODYA can open Ca2+-activated K+ channels (11, 53). This would have the direct opposite effect on the membrane potential compared with clotrimazole, further substantiating the conclusion that the common feature of these drugs, i.e., their ability to inhibit cytochrome P-450 enzymes, is responsible for attenuating increases in permeability.

Metabolism of AA by cytochrome P-450 epoxygenases results in the production of four regioisomers of EETs, 5,6-, 8,9-, 11,12- and 14,15-EET, all of which are produced by canine lungs (42). In this study, addition of 5,6-EET to control lung lobes induced a significant rise in Kf,c. This corresponds well with the data obtained by Graier et al. (17), who showed that 5,6-EET can activate Ca2+ entry into endothelial cells in the absence of store depletion. Other EETs were not tested in our study, so it is not known how they would affect pulmonary endothelial permeability. However, Graier et al. found that 8,9-EET does not stimulate CCE, suggesting that this regioisomer, at least, may not increase Kf,c. In rabbit proximal tubule epithelial cells, 5,6-EET was shown to increase [Ca2+]i, whereas the other EETs were far less potent (23), indicating that 5,6-EET may be the most effective permeability-inducing EET. It must be considered that 5,6-EET is a very labile compound, with a reported half-life in aqueous solution of <1 min (31), and it is therefore possible that metabolites of 5,6-EET are mediating the permeability responses. The EETs are converted by epoxide hydrolases to the corresponding dihydroxyeicosatrienoic acids. 5,6-EET can additionally be metabolized by cyclooxygenase to form vasoactive prostaglandins (12, 13). Inhibition of cyclooxygenase by ibuprofen does not, however, modify thapsigargin-induced increases in permeability (data not shown). That is, the increase in Kf,c induced by thapsigargin is neither less nor more when ibuprofen is used as a pretreatment regimen. This suggests that metabolism of EETs by cyclooxygenase does not bias our results and, furthermore, that prostaglandins are not responsible for the effects seen in this study.

Previously, Roy et al. and Ivey et al. demonstrated that, after pacing-induced heart failure, canine lungs become resistant to changes in permeability normally induced in control lungs by ANG II (37) and thapsigargin (21). We hypothesized that 5,6-EET production may be diminished in the pulmonary endothelium of these lung lobes such that CCE is not activated and there is no rise in permeability. However, addition of exogenous 5,6-EET to lungs from paced animals did not uncover any permeability response. In fact, the epoxygenase metabolite actually induced a decrease in basal permeability. Thus although 5,6-EET can mediate increases in Kf,c in control lungs, it does not appear to be the missing link that renders the lung resistant to injury after the development of pacing-induced heart failure. It seems more likely that the permeability mechanism sensitive to increases in 5,6-EET becomes unresponsive after pacing. Obviously, further experiments are needed to determine exactly what changes are occurring in these lungs such that they fail to respond to permeability-inducing agonists.

The EETs are known vasodilators in various vascular beds including the cerebral microcirculation of the cat and rat (12) and the rat intestinal microvasculature (31). Furthermore, 5,6-EET has been demonstrated to relax preconstricted canine coronary arterial and pulmonary venous rings (36, 42). In the present study, however, addition of 5,6-EET to the lung perfusate had no effect on Ra or Rv in either control or paced lobes. The reason for the difference in our results is unclear, although it may be due partly to discrepancies between species and/or vascular beds or may simply be due to our use of intact lung lobes with minimal vascular tone. The difference in the hemodynamic effects of 5,6-EET and thapsigargin might, at first glance, appear to be discrepant given our discussion of the involvement of cytochrome P-450 metabolites in thapsigargin-induced permeability changes. However, one should consider that thapsigargin can stimulate Ca2+ influx in a number of cell types, including smooth muscle (16), and so can elicit vasoconstriction directly. In contrast, although 5,6-EET is known to increase Ca2+ influx in endothelial cells (17), it does not do so in smooth muscle (16).

The results in this study suggest that cytochrome P-450-mediated metabolism of AA is important in the signaling pathway triggering CCE in pulmonary endothelium. Other investigators have produced data in different cell types that support this hypothesis. Törnquist et al. (44) showed that stimulation of rat thyroid FRTL-5 cells with thapsigargin results in an activation of PLA2 and a subsequent release of AA. More interestingly, cytochrome P-450 inhibitors, including clotrimazole, reduced thapsigargin-induced Ca2+ entry in a dose-dependent manner. Similarly, stimulation of CCE in rat thymocytes is attenuated in the presence of cytochrome P-450 inhibitors (2). In our isolated lung model, administration of ANG II, which stimulates Ins(1,4,5)P3 production and thus CCE, increases endothelial permeability in control lungs (37). In rabbit proximal tubule cells, ANG II was shown to stimulate the production of 5,6-EET; exogenous 5,6-EET and ANG II similarly increased [Ca2+]i; and Ca2+ transients induced by ANG II were blocked by cytochrome P-450 inhibitors (23). These observations provide further evidence that cytochrome P-450 enzymes are involved in the CCE pathway.

The validity of our conclusions relies on the specificity of our measurement of microvascular permeability. Kf,c evaluates the hydraulic conductance of the entire pulmonary microvasculature and therefore provides information regarding the state of the endothelial barrier alone, thus allowing us to differentiate between smooth muscle- and endothelial-mediated events. This is extremely useful when monitoring the effects of agents such as thapsigargin, which induce a rise in pulmonary vascular resistance. Previously, Rippe et al. (35) and Townsley and colleagues (47, 48) found that a marked vasoconstriction induced by various agonists such as norepinephrine, histamine, serotonin, and AA does not influence Kf,c. This point is further validated by the fact that lung lobes from paced animals have an elevated vasoconstrictor response to thapsigargin compared with control lobes, but the drug still fails to modify permeability (21).

Finally, one caveat to our conclusion that endothelium- derived cytochrome P-450 metabolites of AA are involved in thapsigargin-mediated increases in endothelial permeability is that our results could be indirect due to the effects of these drugs on cells other than endothelium. Thapsigargin is known to stimulate Ca2+ influx in neutrophils (26), and imidazole antimycotics such as clotrimazole have been shown to decrease agonist- or thapsigargin-stimulated Ca2+ influx in both neutrophils and platelets (26, 38). In addition, platelets at least are known to synthesize both EETs and 20-HETE from AA (52), although only 20-HETE has been reported from neutrophils (20). Thus, in our blood-perfused lobes, it is possible that thapsigargin stimulates release of secretory products such as platelet-activating factor or oxygen radicals from either platelets or neutrophils and that inhibition of such by cytochrome P-450 inhibitors could potentially explain our results. Although possible, we believe that this scenario is unlikely. For example, clotrimazole has been shown to inhibit phorbol ester-induced release of superoxide anions from neutrophils (19). However, this effect required doses of clotrimazole severalfold higher than that used in the present study. Furthermore, pretreatment with oxygen radical scavengers does not prevent thapsigargin-induced increases in microvascular permeability in buffer-perfused rat lungs (7), a model where neutrophils at least are known to be retained in the lung. In addition, platelet-activating factor, a secretory product of neutrophils and platelets, does not increase endothelial permeability in blood-perfused canine lung (41). Finally, in contrast to the stimulatory effect of 5,6-EET on Ca2+ influx in endothelial cells (17), EETs have been shown to inhibit thapsigargin-induced entry of extracellular Ca2+ in platelets (24). We are not aware of any study in which the effect of EETs on neutrophil function has been evaluated. Collectively, these observations argue against an indirect role for cytochrome P-450-mediated signaling in the thapsigargin permeability response in the canine lung. Nonetheless, our conclusions regarding these results are suggestive rather than definitive.

In summary, results of this study show that the epoxygenase metabolite 5,6-EET can increase pulmonary microvascular permeability in normal, blood-perfused canine lungs. Furthermore, inhibition of the formation of EETs with two distinct cytochrome P-450 inhibitors attenuates the marked rise in permeability induced by thapsigargin. Our data therefore suggest that metabolism of AA via cytochrome P-450 epoxygenases is involved in pathways mediating pulmonary endothelial permeability and may provide a signaling mechanism between Ca2+ store depletion and Ca2+ entry via the plasma membrane SOC in pulmonary endothelial cells. Finally, although addition of 5,6-EET increases permeability in control lung lobes, this effect is not seen after pacing-induced heart failure, suggesting that the resistance to injury observed in these lobes is not due to a deficiency in 5,6-EET production.

    ACKNOWLEDGEMENTS

We thank Dr. D. Lynn Dyess and Jimmy Lakey for invaluable assistance.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-39045.

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. §1734 solely to indicate this fact.

Address for reprint requests: C. Ivey, Dept. of Physiology, MSB 3024, Univ. of South Alabama, Mobile, AL 36688.

Received 2 March 1998; accepted in final form 29 May 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Alvarez, J., M. Montero, and J. Garcia-Sancho. Cytochrome P-450 may link intracellular Ca2+ stores with plasma membrane Ca2+ influx. Biochem. J. 274: 193-197, 1991[Medline].

2.   Alvarez, J., M. Montero, and J. Garcia-Sancho. Cytochrome P450 may regulate plasma membrane Ca2+ permeability according to the filling state of the intracellular Ca2+ stores. FASEB J. 6: 786-792, 1992[Abstract/Free Full Text].

3.   Alvarez, J., M. Montero, and J. Garcia-Sancho. High affinity inhibition of Ca2+-dependent K+ channels by cytochrome P-450 inhibitors. J. Biol. Chem. 267: 11789-11793, 1992[Abstract/Free Full Text].

4.   Berridge, M. J. Inositol trisphosphate and calcium signalling. Nature 361: 315-325, 1993[Medline].

5.   Blakney, G. B., and A. J. Dinwoodie. A spectrophotometric scanning technique for the rapid determination of plasma hemoglobin. Clin. Biochem. 8: 96-102, 1975[Medline].

6.   Brugnara, C., L. de Franceschi, and S. L. Alper. Inhibition of Ca2+-dependent K+ transport and cell dehydration in sickle erythrocytes by clotrimazole and other imidazole derivatives. J. Clin. Invest. 92: 520-526, 1993[Medline].

7.   Chetham, P. M., H. A. Guldemeester, N. Mons, G. H. Brough, J. P. Bridges, W. J. Thompson, and T. Stevens. Ca2+-inhibitable adenylyl cyclase and pulmonary microvascular permeability. Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17): L22-L30, 1997[Abstract/Free Full Text].

8.   Corey, E. J., H. Niwa, and J. R. Falck. Selective epoxidation of eicosa-cis-5,8,11,14-tetraenoic (arachidonic) and eicosa-cis-8,11,14-trienoic acid. J. Am. Chem. Soc. 101: 1586-1587, 1979.

9.   Davies, E. V., and M. B. Hallett. A soluble cellular factor directly stimulates Ca2+ entry in neutrophils. Biochem. Biophys. Res. Commun. 206: 348-354, 1995[Medline].

10.   Dolor, R. J., L. M. Hurwitz, Z. Mirza, H. C. Strauss, and A. R. Whorton. Regulation of extracellular Ca2+ entry in endothelial cells: role of intracellular Ca2+ pool. Am. J. Physiol. 262 (Cell Physiol. 31): C171-C181, 1992[Abstract/Free Full Text].

11.   Edwards, G., P. M. Zygmunt, E. D. Hogestatt, and A. H. Weston. Effects of cytochrome P450 inhibitors on potassium currents and mechanical activity in rat portal vein. Br. J. Pharmacol. 119: 691-701, 1996[Abstract].

12.   Ellis, E. F., R. J. Police, L. Yancey, J. S. McKinney, and S. C. Amruthesh. Dilation of cerebral arterioles by cytochrome P-450 metabolites of arachidonic acid. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H1171-H1177, 1990[Abstract/Free Full Text].

13.   Fitzpatrick, F. A., and R. C. Murphy. Cytochrome P-450 metabolism of arachidonic acid: formation and biological actions of "epoxygenase"-derived eicosanoids. Pharmacol. Rev. 40: 229-241, 1988[Medline].

14.   Garcia, J. G., and K. L. Schaphorst. Regulation of endothelial cell gap formation and paracellular permeability. J. Investig. Med. 43: 117-126, 1995[Medline].

15.   Goeckeler, Z. M., and R. B. Wysolmerski. Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization and myosin phosphorylation. J. Cell Biol. 130: 613-627, 1995[Abstract].

16.   Graber, M. N., A. Alfonso, and D. L. Gill. Recovery of Ca2+ pools and growth in Ca2+ pool-depleted cells is mediated by specific epoxyeicosatrienoic acids derived from arachidonic acid. J. Biol. Chem. 272: 29546-29553, 1997[Abstract/Free Full Text].

17.   Graier, W. F., S. Simecek, and M. Sturek. Cytochrome P450 mono-oxygenase-regulated signalling of Ca2+ entry in human and bovine endothelial cells. J. Physiol. (Lond.) 482: 259-274, 1995[Abstract].

18.   Harder, D. R., W. B. Campbell, and R. J. Roman. Role of cytochrome P-450 enzymes and metabolites of arachidonic acid in the control of vascular tone. J. Vasc. Res. 32: 79-92, 1995[Medline].

19.   Hegemann, L., G. F. Webster, and K. Wolff. Selective calmodulin antagonists fail to inhibit phorbol ester-induced superoxide anion release from human neutrophils: effects of antifungal azole derivatives. Br. J. Dermatol. 135: 199-203, 1996[Medline].

20.   Hill, E., and R. C. Murphy. Quantitation of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) produced by human polymorphonuclear leukocytes using electron capture ionization gas chromatography/mass spectrometry. Biol. Mass Spectrom. 21: 249-253, 1992[Medline].

21.   Ivey, C. L., B. J. Roy, and M. I. Townsley. Ablation of lung endothelial injury after pacing-induced heart failure is related to alterations in calcium signaling. Am. J. Physiol. 275 (Heart Circ. Physiol. 44): H844-H851, 1998[Abstract/Free Full Text].

22.   Linehan, J. H., C. A. Dawson, and D. A. Rickaby. Distribution of vascular resistance and compliance in a dog lung lobe. J. Appl. Physiol. 53: 158-168, 1982[Abstract/Free Full Text].

23.   Madhun, Z. T., D. A. Goldthwait, D. McKay, U. Hopfer, and J. G. Douglas. An epoxygenase metabolite of arachidonic acid mediates angiotensin II-induced rises in cytosolic calcium in rabbit proximal tubule epithelial cells. J. Clin. Invest. 88: 456-461, 1991[Medline].

24.   Malcolm, K. C., and F. A. Fitzpatrick. Epoxyeicosatrienoic acids inhibit Ca2+ entry into platelets stimulated by thapsigargin and thrombin. J. Biol. Chem. 267: 19854-19858, 1992[Abstract/Free Full Text].

25.   Marrero, M. B., W. G. Paxton, J. L. Duff, B. C. Berk, and K. E. Bernstein. Angiotensin II stimulates tyrosine phosphorylation of phospholipase C-gamma 1 in vascular smooth muscle cells. J. Biol. Chem. 269: 10935-10939, 1994[Abstract/Free Full Text].

26.   Montero, M., J. Garcia-Sancho, and J. Alvarez. Comparative effects of cytochrome P-450 inhibitors on Ca2+ and Mn2+ entry induced by agonists or by emptying the Ca2+ stores of human neutrophils. Biochim. Biophys. Acta 1177: 127-133, 1993[Medline].

27.   Moy, A. B., S. S. Shasby, B. Scott, and D. M. Shasby. The effect of histamine and cyclic adenosine monophosphate on myosin light chain phosphorylation in human umbilical vein endothelial cells. J. Clin. Invest. 92: 1198-1206, 1993[Medline].

28.   Moy, A. B., J. Van Engelenhoven, J. Bodmer, J. Kamath, C. Keese, I. Giaever, S. Shasby, and D. M. Shasby. Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces. J. Clin. Invest. 97: 1020-1027, 1996[Abstract/Free Full Text].

29.   Parekh, A. B., H. Terlau, and W. Stuhmer. Depletion of InsP3 stores activates a Ca2+ and K+ current by means of a phosphatase and a diffusable messenger. Nature 364: 814-818, 1993[Medline].

30.   Pearce, M. L., J. Yamashita, and J. Beazell. Measurement of pulmonary edema. Circ. Res. 16: 482-488, 1965.

31.   Proctor, K. G., J. R. Falck, and J. Capdevila. Intestinal vasodilation by epoxyeicosatrienoic acids: arachidonic acid metabolites produced by a cytochrome P450 monoxygenase. Circ. Res. 60: 50-59, 1987[Abstract].

32.   Putney, J. W., Jr. A model for receptor-regulated calcium entry. Cell Calcium 7: 1-12, 1986[Medline].

33.   Putney, J. W., Jr., J. Poggioli, and S. J. Weiss. Receptor regulation of calcium release and calcium permeability in parotid gland cells. Philos. Trans. R. Soc. Lond. B Biol. Sci. 296: 37-45, 1981[Medline].

34.   Randriamampita, C., and R. Y. Tsien. Emptying of intracellular Ca2+ stores releases a novel small messenger that stimulates Ca2+ influx. Nature 364: 809-814, 1993[Medline].

35.   Rippe, B., R. C. Allison, J. C. Parker, and A. E. Taylor. Effects of histamine, serotonin, and norepinephrine on circulation of dog lungs. J. Appl. Physiol. 57: 223-232, 1984[Abstract/Free Full Text].

36.   Rosolowsky, M., J. R. Falck, J. T. Willerson, and W. B. Campbell. Synthesis of lipoxygenase and epoxygenase products of arachidonic acid by normal and stenosed canine coronary arteries. Circ. Res. 66: 608-621, 1990[Abstract].

37.   Roy, B. J., V. H. Pitts, and M. I. Townsley. Pulmonary vascular response to angiotensin II in canine pacing-induced heart failure. Am. J. Physiol. 271 (Heart Circ. Physiol. 40): H222-H227, 1996[Abstract/Free Full Text].

38.   Sargeant, P., R. W. Farndale, and S. O. Sage. The imidazole antimycotics econazole and miconazole reduce agonist-evoked protein-tyrosine phosphorylation and evoke membrane depolarization in human platelets: cautions for their use in studying Ca2+ signalling pathways. Cell Calcium 16: 413-418, 1994[Medline].

39.   Schilling, W. P., O. A. Cabello, and L. Rajan. Depletion of the inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ store in vascular endothelial cells activates the agonist-sensitive Ca2+-influx pathway. Biochem. J. 284: 521-530, 1992[Medline].

40.   Sheldon, R., A. Moy, K. Lindsley, S. Shasby, and D. M. Shasby. Role of myosin light-chain phosphorylation in endothelial cell retraction. Am. J. Physiol. 265 (Lung Cell. Mol. Physiol. 9): L606-L612, 1993[Abstract/Free Full Text].

41.   Shibamoto, T., Y. Yamaguchi, T. Hayashi, Y. Saeki, M. Kawamoto, and S. Koyama. PAF increases capillary pressure but not vascular permeability in isolated blood-perfused canine lungs. Am. J. Physiol. 264 (Heart Circ. Physiol. 33): H1454-H1459, 1993[Abstract/Free Full Text].

42.   Stephenson, A. H., R. S. Sprague, N. L. Weintraub, L. McMurdo, and A. J. Lonigro. Inhibition of cytochrome P-450 attenuates hypoxemia of acute lung injury in dogs. Am. J. Physiol. 270 (Heart Circ. Physiol. 39): H1355-H1362, 1996[Abstract/Free Full Text].

43.   Thastrup, O., P. J. Cullen, B. R. Drobak, M. R. Hanley, A. P. Dawson, and B. Durbak. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc. Natl. Acad. Sci. USA 87: 2466-2470, 1990[Abstract].

44.   Törnquist, K., E. Ekokoski, and L. Forss. Thapsigargin-induced calcium entry in FRTL-5 cells: possible dependence on phospholipase A2 activation. J. Cell. Physiol. 160: 40-46, 1994[Medline].

45.   Townsley, M. I., Z. Fu, O. Mathieu-Costello, and J. B. West. Pulmonary microvascular permeability. Responses to high vascular pressure after induction of pacing-induced heart failure in dogs. Circ. Res. 77: 317-325, 1995[Abstract/Free Full Text].

46.   Townsley, M. I., R. J. Korthuis, B. Rippe, J. C. Parker, and A. E. Taylor. 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].

47.   Townsley, M. I., R. J. Korthuis, and A. E. Taylor. Effects of arachidonate on permeability and resistance distribution in canine lungs. J. Appl. Physiol. 58: 206-210, 1985[Abstract/Free Full Text].

48.   Townsley, M. I., V. H. Pitts, J. L. Ardell, Z. Zhao, and W. H. Johnson, Jr. Altered pulmonary microvascular reactivity to norepinephrine in canine pacing-induced heart failure. Circ. Res. 75: 347-356, 1994[Abstract].

49.   Ullian, M. E., and S. L. Linas. Angiotensin II surface receptor coupling to inositol trisphosphate formation in vascular smooth muscle cells. J. Biol. Chem. 265: 195-200, 1990[Abstract/Free Full Text].

50.   Vaca, L., and D. L. Kunze. Depletion of intracellular Ca2+ stores activates a Ca2+-selective channel in vascular endothelium. Am. J. Physiol. 267 (Cell Physiol. 36): C920-C925, 1994[Abstract/Free Full Text].

51.   Weidner, W. J. Extravascular lung water content in the domestic fowl (Gallus domesticus). Physiol. Zool. 51: 267-271, 1978.

52.   Zhu, Y., E. B. Schieber, J. C. McGiff, and M. Balazy. Identification of arachidonate P-450 metabolites in human platelet phospholipids. Hypertension 25: 854-859, 1995[Abstract/Free Full Text].

53.   Zou, A. P., J. T. Fleming, J. R. Falck, E. R. Jacobs, D. Gebremedhin, D. R. Harder, and R. J. Roman. 20-HETE is an endogenous inhibitor of the large-conductance Ca2+-activated K+ channel in renal arterioles. Am. J. Physiol. 270 (Regulatory Integrative Comp. Physiol. 39): R228-R237, 1996[Abstract/Free Full Text].

54.   Zou, A. P., J. D. Imig, M. Kaldunski, P. R. Ortiz de Montellano, Z. Sui, and R. J. Roman. Inhibition of renal vascular 20-HETE production impairs autoregulation of renal blood flow. Am. J. Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F275-F282, 1994[Abstract/Free Full Text].

55.   Zou, A. P., Y. H. Ma, Z. H. Sui, P. R. Ortiz de Montellano, J. E. Clark, B. S. Masters, and R. J. Roman. Effects of 17-octadecynoic acid, a suicide-substrate inhibitor of cytochrome P450 fatty acid omega -hydroxylases, on renal function in rats. J. Pharmacol. Exp. Ther. 268: 474-481, 1994[Abstract].


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