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Am J Physiol Heart Circ Physiol 294: H1435-H1443, 2008. First published January 18, 2008; doi:10.1152/ajpheart.01115.2007
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Effects of {omega}-hydroxylase product on distal human pulmonary arteries

Caroline Morin,1 Christelle Guibert,4 Marco Sirois,2 Vincent Echave,2 Marcio M. Gomes,3 and Eric Rousseau1

1Le Bilarium, Department of Physiology and Biophysics, 2Service of Thoracic Surgery, and 3Department of Pathology, Université de Sherbrooke, Sherbrooke, Quebéc, Canada; and 4Laboratoire de Physiologie Cellulaire Respiratoire, Institut National de la Santé et de la Recherche Médicale U885, Université Bordeaux 2, Bordeaux, France

Submitted 25 September 2007 ; accepted in final form 7 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The aim of the present study was to provide a mechanistic insight into how 20-hydroxyeicosatetraenoic acid (20-HETE) relaxes distal human pulmonary arteries (HPAs). This compound is produced by {omega}-hydroxylase from free arachidonic acid. Tension measurements, performed on either fresh or 1 day-cultured pulmonary arteries, revealed that the contractile responses to 1 µM 5-hydroxytryptamine were largely relaxed by 20-HETE in a concentration-dependent manner (0.01–10 µM). Iberiotoxin pretreatments (10 nM) partially decreased 20-HETE-induced relaxations. However, 10 µM indomethacin and 3 µM SC-560 pretreatments significantly reduced the relaxations to 20-HETE in these tissues. The relaxing responses induced by the eicosanoid were likely related to a reduced Ca2+ sensitivity of the myofilaments since free Ca2+ concentration ([Ca2+])-response curves performed on β-escin-permeabilized cultured explants were shifted toward higher [Ca2+]. 20-HETE also abolished the tonic responses induced by phorbol-ester-dibutyrate (a PKC-sensitizing agent). Western blot analyses, using two specific primary antibodies against the PKC-potentiated inhibitory protein CPI-17 and its PKC-dependent phosphorylated isoform pCPI-17, confirmed that 20-HETE interferes with this intracellular process. We also investigated the effect of 20-HETE on the activation of Rho-kinase pathway-induced Ca2+ sensitivity. The data demonstrated that 20-HETE decreased U-46619-induced Ca2+ sensitivity on arteries. Hence, this observation was correlated with an increased staining of p116Rip, a RhoA-binding protein. Together, these results strongly suggest that the 20-hydroxyarachidonic acid derivative is a potent modulator of tone in HPAs in vitro.

20-hydroxyeicosatetraenoic acid; calcium sensitivity; tension measurement

PULMONARY ARTERY VASOCONSTRICTION and vascular remodeling greatly contribute to a sustained elevation of pulmonary vascular resistance and pulmonary arterial pressure in patients with pulmonary arterial hypertension (PAH), an often fatal hemodynamic abnormality (11). Pulmonary vasoconstriction is believed to be an early component of the pulmonary hypertensive process and has been attributed to an impaired production of vasodilators [nitric oxide (NO) and prostacyclin] and an increased production of vasoconstrictors (endothelin-1 and serotonin) (11). In that respect, vasodilators have some beneficial effects on PAH. Among various existing pulmonary arterial vasodilators, such as NO, sildenafil, and cGMP (24, 25), 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P-450 4A metabolite of arachidonic acid, consistently dilates pulmonary arteries in several species, including humans (2). 20-HETE induces a relaxation in bovine pulmonary arteries, whereas it induces a contraction in bovine renal arteries (12, 15), suggesting a specific mode of action of 20-HETE according to vascular beds. Interestingly, on one hand, hypoxia blocks the conversion of arachidonic acid into 20-HETE in rabbit lung (34), whereas, on the other hand, chronic hypoxia induces sustained elevated pulmonary arterial pressure and vascular remodeling leading to PAH (26), suggesting a potential role for a lower {omega}-hydroxylase activity and a decreased production of endogenous 20-HETE in chronic hypoxia-induced PAH. Our hypothesis is strengthened in that cytochrome P-450 4A ({omega}-hydroxylase) protein levels were decreased in a model of shunt-induced PAH in piglets (23). Our understanding of the mechanisms involved in the pulmonary arterial dilation, and especially the 20-HETE-induced relaxation, thus appears to be of great interest in the new therapeutic development for PAH.

Human and rabbit lung microsomes are able to metabolize arachidonic acid into 20-HETE, possibly via the activation of cytochrome P-450 4A, which is widely distributed in peripheral lung tissues, including small and large pulmonary arteries and airways as well (2, 35). Such a wide distribution of the cytochrome P-450 4A raises the possibility that 20-HETE generated from nonvascular tissue could serve as an endogenous and paracrine modulator of pulmonary vascular tone (2, 35). Although arachidonic acid-induced relaxation in human pulmonary arteries (HPAs) involves cytochrome P-450-dependent metabolites and potassium channels (9), the precise molecular mode of action of 20-HETE remains to be ascertained. Since 20-HETE may be of putative clinical interest for PAH (11, 24, 25), we have focused the present study on the alternative mechanisms involved in the 20-HETE-induced relaxation in HPAs.

The aim of the present work was to assess the physiological effects of 20-HETE on resistance vessels. Complementary approaches used include 1) tension measurements of relaxation on human arterial rings; 2) analyses of the effects of 20-HETE on the Ca2+ sensitivity of the mechanical apparatus; and 3) evaluation of the status and expression of the PKC-potentiated inhibitory protein CPI-17 and p116Rip proteins, which are involved in the control of the Ca2+ sensitivity in vascular smooth muscle (VSM) cells (17, 30). We report herein the first evidence that the iberiotoxin (IbTx) sensitivity of the 20-HETE induces concentration-dependent relaxations, and we demonstrate that 20-HETE decreases the Ca2+ sensitivity of myofilaments from distal HPAs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation and culture of human pulmonary tissues. The study was approved by the Ethics Committee (Protocol number CRC 05-088) of our institution, and consent was obtained from each subject. Human lung tissues were obtained from 15 patients undergoing surgery for lung carcinoma. After lobectomy and transport in sterile physiological saline solution, lung samples, distant from the malignant lesion, were dissected by the pathologist. The absence of tumoral infiltration was retrospectively established in all tissue sections by pathological analysis. Tissue samples were immediately placed in Krebs solution containing (in mM) 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, and 11.1 glucose, previously bubbled with 95% O2–5% CO2 (pH 7.4) at 22°C and then immediately transported to a level-two culture room. After the removal of connective tissues, paired rings of similar weight and length (inner diameter, 0.5–0.8 mm) were microdissected from the same pulmonary artery segment. Arterial rings were used as fresh tissue or cultured in individual wells of 24-well culture plates containing DMEM-F12 culture medium (1 ml/well) supplemented with 0.3% penicillin (100 IU/ml) and streptomycin (0.1 mg/ml). Culture plates were placed in a humidified incubator at 37°C under 5% CO2. Explants were maintained in culture for 18 h as previously reported (10).

Isometric tension measurements. The mechanical effects induced by specific agonists and eicosanoids were measured as previously reported (19). Arterial rings were mounted in isolated organ baths, containing 6 ml of Krebs solution at 37°C, and gassed continually with the 95% O2–5% CO2 mixture, to which an initial load of 0.8 g was applied. Tissues were allowed to equilibrate for 1 h in Krebs solution and washed out every 15 min. Passive and active tensions were assessed using transducer systems (Radnoti Glass, Monrovia, CA) coupled to Polyview software (Grass Astro Med, West Warwick, RI) for facilitating data acquisition and analysis.

Permeabilization with β-escin. Arterial rings were mounted in organ baths and stretched to 0.8 g. After the contraction elicited by 1 µM phenylephrine (PE) and/or 5-hydroxytryptamine (5-HT) in normal Krebs solution was measured, the rings were incubated for 20 min in low free Ca2+ relaxing solution containing (in mM) 87 KCl, 5.1 MgCl2, 5.2 NaATP, 10 creatine phosphate, 2 EGTA, and 10 PIPES, brought to a pH of 7.2 with KOH at 23°C, followed by treatment with 50 µM β-escin in relaxing solution for 35 min at 23°C. Ca2+ stores were depleted by the addition of 10 µM A-23187 (19). The arterial rings were washed several times with fresh relaxing solution containing 10 mM EGTA. Calmodulin (1 µM) was present in the bathing solutions throughout the experiments to prevent an alteration of a Ca2+-induced contraction. Tension developed by permeabilized tissue was measured in activating solutions containing 10 mM EGTA and specified concentrations of CaCl2 to yield the desired free Ca2+ concentration, pCa = –log [Ca2+] (20). Step increases in free Ca2+ (pCa = 9.0 to 5.3) were used to induce reproducible concentration-dependent tension response curves, indicating a successful permeabilization of tissues under these conditions. Arterial rings were mounted in isolated organ bath systems, untreated (as control) or treated for 15 min with 100 nM 20-HETE alone, 1 µM phorbol 12,13-dibutyrate (PDBu) combined with 100 nM 20-HETE, or 0.1 µM U-46619 combined, or not, with 100 nM 20-HETE, before the addition of CaCl2 increments.

SDS-PAGE and Western blot analysis. HPAs were dissected, weighed, and promptly transferred in a buffer containing 0.3 M sucrose, 20 mM K-PIPES, 4 mM EGTA, and a cocktail of protease inhibitors (protease-inhibitor pellets from Roche Diagnostics, Indianapolis, IN). Tissues were then homogenized on ice, frozen in liquid nitrogen, and stored at –80°C. Arterial explants were maintained in culture as untreated (control) or treated explants for 18 h with 100 nM 20-HETE alone, 1 µM PDBu combined with 100 nM 20-HETE, or 0.1 µM U-46619 combined with 100 nM 20-HETE before arterial tissue homogenization. For SDS-PAGE, protein samples (20 µg of protein/well) from homogenate fractions were dissolved in 2% SDS and separated on 15% SDS-PAGE using a 3% stacking gel. Gels were cast into a mini-protean III dual cell (Bio-Rad, Mississauga, ON, Canada). Western blot analyses using specific antibodies against CPI-17, phosphorylated (p)CPI-17, p116Rip, and β-actin proteins were performed on homogenate fractions (19). The separated proteins from SDS-PAGE were electrophoretically transferred onto nitrocellulose membranes (Bio-Rad). Transferred membranes were blocked with Tris-buffered solution containing 0.1% Tween 20 (TBS-T) plus 5% nonfat diet milk overnight and then incubated for 180 min with 1 µg/ml of the selected specific antibody in TBS-T. After three washes in TBS-T as above, membranes were incubated for 1 h at 23°C with peroxidase-conjugated donkey anti-rabbit IgG1 antiserum (Amersham, Baie d'Urfé, QC, Canada) and revealed by enhanced chemiluminescence (Roche).

Drugs and chemical reagents. 20-HETE, U-46619, and 14,15-epoxyeicosatrienoic acid (EET) were obtained from Cayman Chemical (Ann Arbor, MI), dissolved in 100% ethanol (EtOH), and stored as 1 mM stock solutions. PDBu and IbTx were purchased from Calbiochem-VWR (Montreal, QC, Canada). The vehicle was tested separately at the maximal concentration used in the presence of an active compound. 5-HT, PE, and indomethacin (Indo) were purchased from Sigma (St. Louis, MO). DMEM-F12 and penicillin-streptomycin were purchased from Gibco Invitrogen (Burlington, ON, Canada).

Data analysis and statistics. Results are expressed as means ± SE; n indicates the number of experiments. Statistical analyses were performed using Student's t-test or a one-way ANOVA. Differences were considered significant when P < 0.05. Data curve fittings were performed using Sigma Plot 9.0 (SPSS-Science, Chicago, IL) to determine EC50 and IC50 values (19).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
20-HETE relaxes HPAs. Tension measurements were performed on distal pulmonary artery rings to test the effect of 20-HETE on resting tone. Tissues were subjected to 0.8 g basal tone. Cumulative concentrations of 20-HETE (0.01–10 µM) resulted in concentration-dependent relaxing effects (Fig. 1A) with an IC50 value of 0.33 µM. The vehicle, ethanol, had no significant effect on the resting tone. 20-HETE (1 µM) yielded a mean relaxation of 1.68 ± 0.19 mN on these HPAs. The effects of 20-HETE were then assessed on pulmonary arteries precontracted with 1 µM 5-HT. The addition of cumulative concentrations of 20-HETE resulted in concentration-dependent relaxing effects on the active tone (Fig. 1B). Figure 1C quantifies the mean concentration-dependent relaxing effects induced by 20-HETE on 1 µM 5-HT and 1 µM PE precontracted arteries, with IC50 values of 0.22 ± 0.03 and 0.36 ± 0.03 µM, respectively.


Figure 1
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  		Fig. 1. 20-Hydroxyeicosatetraenoic acid (HETE)-induced concentration-dependent relaxations of human pulmonary artery (HPA). A: quantitative analysis of the relaxing responses induced by cumulative addition of 20-HETE on resting tone from distal pulmonary arteries (n = 14 experiments). B: typical recording of the relaxing response induced by 20-HETE on pulmonary artery precontracted with 1 µM 5-hydroxytryptamine (5-HT). C: quantitative analysis of the concentration-dependent relaxing responses induced by 20-HETE on distal pulmonary arteries constricted with either 1 µM 5-HT or 1 µM phenylephrine (PE). Points represent means ± SE with n = 16 experiments and n = 14 experiments for each experimental condition, respectively.

 
Endothelium removal and cyclooxygenase inhibition modified 20-HETE responses. Tension measurements were performed on endothelium-denuded pulmonary arteries to test the effect of 20-HETE on precontracted arterial rings. Figure 2A displays the relaxing effects of 1 µM 20-HETE on both intact (control, left) and endothelium-denuded (right) HPA rings precontracted to 1 µM 5-HT. In the absence of an endothelial layer, 1 µM 20-HETE relaxed 5-HT-precontracted HPA; however, the relaxation was largely inhibited (–71%). Since it was previously reported that the effects of 20-HETE can be totally inhibited in the presence of 1 µM Indo, a nonselective cyclooxygenase (COX) inhibitor in rabbit pulmonary arteries (14), the relaxing effects of 20-HETE were assessed in the absence and presence of Indo. Quantitative analysis demonstrated that the relaxations induced by 20-HETE were largely reduced by 10 µM Indo and 3 µM SC-560, a specific COX-1 inhibitor (Fig. 2B), thus indicating that the relaxing response to 20-HETE is likely modulated by an intracellular COX-derived prostanoid metabolite (5). Consequently, we have assessed the putative effects of 20-hydroxy-PGE2 on tone. This compound induced a low level of relaxation (11% at 1 µM) on 5-HT-precontracted arteries (Fig. 2C), suggesting that a different metabolite was produced.


Figure 2
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  		Fig. 2. Effects of endothelium removal, cyclooxygenase (COX) inhibition, and N-methylsulphonyl-12,12-dibromododec-11-enamide (DDMS) pretreaments on 20-HETE responses. A: paired recordings of relaxing responses induced by 1 µM 20-HETE on control and endothelium-denuded HPA precontracted with 1 µM 5-HT. B: quantitative analysis of the relaxing responses to 1 µM 20-HETE on HPA constricted with 1 µM 5-HT before (control) and after pretreatment with either 10 µM indomethacin (Indo) or 3 µM SC-560. The latter was used as a selective COX-1 inhibitor (n = 15 experiments). *P < 0.05 was considered as significant by ANOVA. C: bar histogram demonstrating the limited relaxing effect of 20-OH-PGE2 on HPA compared with the 20-HETE relaxing responses (n = 12 experiments for each condition). D: paired recordings of responses generated by 1 µM PE before (control) and after 3 µM DDMS pretreatment. DDMS was used as a cytochrome P-450 (CYP450) {omega}-hydroxylase inhibitor. E: Western blot analyses, using specific antibodies against cytochrome P-450 4A (CYP4A) and β-actin, were performed on 3 distinct homogenates. The CYP450 4A {omega}-hydroxylase isoform was detected in all HPA fractions tested (n = 5 experiments).

 
Endogenous production of 20-HETE. The specific cytochrome P-450 {omega}-hydroxylase inhibitor N-methylsulphonyl-12,12-dibromododec-11-enamide (DDMS) (21) was used to assess the putative role of the endogenous production of 20-HETE. When compared with the control response to 1 µM PE, pretreatment with 3 µM DDMS had basically no effect on basal tone but significantly increased the tonic responses to 1 µM PE by 36%, as illustrated in Fig. 2D. Thus cytochrome P-450 {omega}-hydroxylase inhibition with DDMS, which eventually lowered the endogenous production of 20-HETE in fresh HPAs, resulted in an increased PE-induced tension, which might be a relevant observation in PAH. Moreover, Western blot analysis of a distinct homogenate derived from fresh, 12-, and 24-h cultured HPA rings revealed an immunostaining of a protein band at 52 kDa. This single band would be compatible with the presence of cytochrome P-450 4A (CYP4A), the enzyme that produced 20-HETE from arachidonic acid, in all tested fractions (Fig. 2E). Note that the β-actin staining was constant from one preparation to the other.

Involvement of large-conductance Ca2+-activated K+ channels and effect of 20-HETE on membrane potential of HPA smooth muscle cells. Since it has recently been reported that 20-HETE activates large-conductance Ca2+-activated K+ (BKCa) channels in human airway smooth muscle (ASM) (20), the relaxing effects of 20-HETE on HPA were assessed in the absence and presence of 10 nM IbTx, a specific BKCa channels blocker (1). Figure 3A shows two sequential recordings in which IbTx pretreatment did not modify the contractile response to 1 µM 5-HT but did result in a significant inhibition (30%) of the amplitude and rate of relaxation induced by 20-HETE compared with the control response. Thus preincubation with 10 nM IbTx had a partial inhibitory effect on the relaxation induced by 3 µM 20-HETE, suggesting that the activation of BKCa channels would only mediate part of the responses induced by this eicosanoid in HPAs.


Figure 3
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  		Fig. 3. Effects of 20-HETE on iberiotoxin (IbTx)-pretreated tissue and on membrane potential of HPA smooth muscle cells. A: paired recordings of relaxing responses induced by 3 µM 20-HETE before (control) and after 10 nM IbTx pretreatment on HPA precontracted with 1 µM 5-HT (n = 16 experiments). B: recording of the membrane potential under resting condition and following addition of 3 µM 20-HETE. At the end of each recording, the microelectrode was removed from the smooth muscle cell to validate the measurement. C: mean resting membrane potential values determined for 3 µM 20-HETE on HPA smooth muscle cells. Note that in the presence of 10 nM IbTx (pretreatment), the mean membrane potential value determined after 3 µM 20-HETE addition was similar to the mean control value (n = 8 experiments for each experimental condition, *P < 0.05 was considered as significant by ANOVA).

 
The effects of 20-HETE on VSM cell membrane potential were verified following microelectrode impalement on HPA explants. Figure 3B illustrates a representative recording of the hyperpolarizing effects induced upon the addition of 3 µM 20-HETE from a resting membrane potential of –51 mV. At the end of each experiment, the microelectrode was removed from the VSM cell to validate the recordings. The mean electrophysiological effects of 20-HETE on HPA tissues are shown in Fig. 3C. A hyperpolarizing effect of –7.2 ± 0.8 mV was recorded upon the addition of 3 µM 20-HETE. After 10 nM IbTx pretreatments, the mean membrane potential value determined upon the 3 µM 20-HETE addition was –50.4 ± 0.9 mV, which was not significantly different from the mean control value (–49.5 ± 1 mV).

20-HETE partially relaxes K+-induced tension. Experiments were designed to assess the putative effects of 20-HETE on K+-induced tension. Figure 4A displays superimposed representative recordings of the contraction induced by 80 mM KCl on HPAs in control condition (gray line) and following the effects induced by the cumulative addition of 20-HETE on arterial rings from the same lung. Note the slow and concentration-dependent effects of the eicosanoid, which largely differs from the control trace. Quantitative data analysis shows that 20-HETE induced a concentration-dependent relaxing effect on arterial rings precontracted with 80 mM KCl (Fig. 4B). 20-HETE (3 µM) yields a mean relaxation of 62% of the maximal tension evoked by a KCl addition (Fig. 4B). These results confirm that the addition of 20-HETE opposes the processes triggered by KCl, which usually involve membrane depolarization, an activation of voltage-operated Ca2+ channels, Ca2+ entry, a formation of Ca-calmodulin complexes, and an activation of various enzymatic systems, including the myosin light chain (MLC) kinase (MLCK), which phosphorylates the MLCs (16, 30).


Figure 4
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  		Fig. 4. 20-HETE partially relaxes K+ contracture on pulmonary arteries. A: superimposed recordings displaying the tension induced and maintained by 80 mM K+ in control condition (gray line) as well as the relaxing effects induced by cumulative concentrations of 20-HETE on a HPA precontracted with 80 mM K+ (black line). B: quantitative analysis of the mean relaxing responses induced by cumulative concentrations of 20-HETE on HPA. Points represent means ± SE with n = 16 experiments.

 
Effect of 20-HETE on Ca2+ sensitivity. Comparative analyses were performed on β-escin-permeabilized preparations to assess the effect of 20-HETE on the Ca2+ sensitivity of the contractile apparatus. Figure 5A displays superimposed recordings induced by cumulative free [Ca2+] increments in control condition and 1 µM 20-HETE acutely pretreated pulmonary arteries. After 15 min, 20-HETE had a consistent inhibitory effect on Ca2+-dependent tension. The cumulative concentration response curve to free Ca2+ concentrations on permeabilized arterial rings obtained from control and eicosanoid-treated arteries are shown in Fig. 5B. Data analysis demonstrates that 20-HETE reduced the Ca2+ sensitivity of arterial rings, with a shift in EC50 values toward higher Ca2+ concentrations of 2.29 ± 0.03 compared with 0.51 ± 0.03 µM in controls (Fig. 5B). The mean responses demonstrate that 20-HETE pretreatments decrease the Ca2+ sensitivity of permeabilized HPAs by fourfold.


Figure 5
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  		Fig. 5. Effects of 20-HETE treatment on the Ca2+ sensitivity from permeabilized HPAs. A: superimposed recordings illustrating the tension induced by cumulative increases in free [Ca2+] on β-escin-permeabilized pulmonary arteries in control condition (black line) and following 1 µM 20-HETE pretreatment (gray line). B: cumulative concentration response curve to free [Ca2+] obtained from β-escin-permeabilized pulmonary artery rings in control conditions (bullet, n = 16 experiments) and in the presence of 1 µM 20-HETE ({circ}, n = 18 experiments).*Statistically significant.

 
20-HETE modulates key regulatory proteins. In Fig. 6A, PDBu was used as a direct activator of conventional and novel PKCs to stimulate the PKC/CPI-17 pathway and produce tone increases. Protocols were performed on β-escin-permeabilized explants under Ca2+ clamp conditions (pCa = 6). Quantitative analyses clearly demonstrate that 1 µM 20-HETE treatment significantly decreased the average tension triggered at pCa 6 in the presence of 1 µM PDBu (Fig. 6A). Thus Western blot analysis of homogenates from HPA explants challenged with 20-HETE, PDBu, or PDBu + 20-HETE revealed that CPI-17 was present in all tested fractions, whereas its phosphorylated form was reduced in 20-HETE treatments compared with the control tissue. PDBu was used as a positive control to induce CPI-17 phosphorylation. Combined treatment of HPAs with PDBu and 20-HETE reduced the staining level of the CPI-17 phosphorylated form compared with the PDBu-treated tissues. Note that the detection of the pMLC isoform displayed a similar pattern (Fig. 6B). In contrast, β-actin staining was constant from one preparation to the other.


Figure 6
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  		Fig. 6. Modification of CPI-17 phosphorylation and RhoA-binding protein p116Rip expression in HPAs. A: quantitative analysis of the mean tonic responses to 1 µM phorbol 12,13-dibutyrate (PDBu) in the absence or presence of 1 µM 20-HETE (n = 14 experiments). These results suggest that 20-HETE is able to decrease the Ca2+ sensitivity of the myofilaments induced by PDBu in HPAs. B: Western blot analyses, using specific antibodies against CPI-17, phosphorylated (p)CPI-17, and β-actin, were performed on 4 distinct homogenates. Significant reduction in pCPI-17 staining density ratio was quantified in 20-HETE-treated (1 µM) tissues versus the control. C: bar histogram of the relative responses to 1 µM U-46619 in the absence and presence of 1 µM 20-HETE. The eicosanoid pretreatment displayed a marked inhibitory effect on 1 µM U-46619-induced tension at pCa = 6 (n = 14 experiments). D: Western blot analyses were performed on 4 homogenates using 3 specific antibodies against p116Rip, phosphorylated myosin light chain (pMLC), and β-actin, respectively. These results are representative of 6 similar experiments. An increase in p116Rip density ratio was quantified in 20-HETE-treated tissues compared with control and U-46619-treated tissues (*P < 0.05).

 
Complementary experiments were performed to assess whether 20-HETE was able to modify the Ca2+-sensitivity response generated by the activation of the Rho-kinase pathway in pulmonary arteries. U-46619, a training programme receptor agonist, was used to stimulate this pathway (19). Data analysis demonstrates that 20-HETE pretreatment significantly decreased the active tension generated at pCa 6 upon the addition of 1 µM U-46619 compared with untreated tissue (Fig. 6C). Moreover, Western blot analysis of distinct homogenates derived from human arteries revealed that p116Rip was present in all tested fractions, whereas it was significantly increased in 20-HETE-pretreated fractions compared with control (untreated HPAs). A combined treatment of HPAs with 20-HETE and U-46619 increased the staining level of p116Rip compared with the U-46619-treated tissues alone (Fig. 6D), whereas β-actin staining was constant from one preparation to the other.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this report, we demonstrate that 20-HETE decreases resting tone and relaxes precontracted resistant HPAs. Moreover, this relaxation appears to be related to both the activation of K+ conductance of the surface membrane and a shift in Ca2+ sensitivity of the contractile machinery. This is the first report directly assessing the functional and intracellular effects of this eicosanoid in HPAs. Hence, we have uncovered an endogenous production of 20-HETE, the inhibition of which resulted in an increased active tone after pharmacological challenges with either PE or 5-HT. Thus we propose that an eicosanoid such as 20-HETE would play a regulatory role in the fine tuning of resistant HPAs, with physiological consequences on local blood pressure and lung circulation.

Relaxing effects of 20-HETE in human lung. 20-HETE was found to relax smooth muscle from distal pulmonary arteries. This eicosanoid displays potent relaxing effects on HPAs, with IC50 values in the submicromolar range (0.3 µM) on either resting or active tone. To our knowledge, there are only a few publications concerning the relaxing effects of 20-HETE on precontracted rabbit and HPAs (2, 14). Despite the fact that cytochrome P-450 {omega}-hydroxylase has been identified in various lung tissues (13, 18, 28, 33, 35), a key issue has been to demonstrate the endogenous involvement of 20-HETE in HPAs. In HPA tissue, it is relevant to analyze the expression of proteins, such the cytochrome P-450 isoforms that produce 20-HETE. Western blot analysis performed on a HPA homogenate showed the presence of CYP4A isoform in this tissue. Moreover, it has been reported that the cytochrome P-450 4A isozyme represents a {omega}-hydroxylase, which is responsible for 20-HETE production in several human tissues (8). Consequently, the expression of this isoform could be downregulated in a patient diagnosed with PAH (26). Despite legitimate ethical concerns, it would be of interest to assess the in vivo production of 20-HETE in pathological human lungs. Moreover, using a new pharmacological {omega}-hydroxylase inhibitor such as DDMS (15, 21), which minimizes the endogenous production of 20-HETE, it was possible to amplify tonic responses to various vasoconstrictive agents. This strategy enabled us to evaluate the putative role of this eicosanoid in lung vascular tissues. Hence, it was hypothesized that 20-HETE could play the role of a paracrine mediator in the HPA wall. Its basal production would facilitate PA dilation and help to maintain a low (12–15 mmHg) blood pressure in this specific segment of the vascular apparatus. However, the difference of reactivity to 20-HETE between conduit and resistance arteries has not been addressed yet. In fact, 20-HETE is thought to play an important role in regulating tone in distal HPA. It was recently reported that 20-HETE also induced a complete relaxation of distal human ASM, precontracted with muscarinic or histaminic agonists (20). In contrast, consistent contractions have been measured and reported in rodents such as guinea pig bronchi (4). Nevertheless, the relaxations induced by 20-HETE might be of pharmacological interest in pulmonary hypertension, since it could be used to treat a critical clinical condition, which is known to be refractory to classical treatments.

COX inhibition affects 20-HETE-dependent relaxations in HPA. Since 20-HETE could be metabolized by COX, the existence of several metabolites has been postulated in other vascular beds and lung tissues (13). Moreover, endothelium removal is often difficult to achieve in the preparations used; thus one approach toward minimizing the contribution of the COX pathway is to use specific pharmacological COX inhibitors such as Indo and SC-560. The relaxations induced by 20-HETE were largely reduced by Indo and SC-560, which suggests that the responses to 20-HETE were possibly modulated by an intracellular COX-derived prostanoid, the identity of which remains to be ascertained (5).

Involvement of K+-selective channels. In the present study, IbTx unequivocally abolished 30% of the relaxing effect induced by 20-HETE in 5-HT precontracted vessels, under normal external K+ concentration, which suggests that the eicosanoid activates BKCa channels, which would result in a membrane hyperpolarization. The intracellular microelectrode technique revealed that 20-HETE induced a significant hyperpolarization of VSM cells from HPAs. Because IbTX prevented the hyperpolarizing effects and reduced the relaxation induced by 20-HETE, BKCa channel activation thus appears to be a key determinant in the regulation of HPA tone. Moreover, it was previously shown that 20-HETE induces similar concentration-dependent relaxing effects in native and cultured human bronchi (20). This effect was shown to be clearly related to a hyperpolarization of ASM cells due to the activation of K+ conductance (9). Microelectrode measurements demonstrated that 20-HETE hyperpolarizes human ASM cells and that this effect was abolished by 10 nM IbTx (20). Our current results suggest that HPAs may respond to an exogenous addition of 20-HETE in a similar fashion than human distal bronchi in which there was a marked activation of BKCa channels (20). Thus the mode of action of this compound may be related to its molecular interactions with specific ionic channels of the surface membrane, since it was already demonstrated in guinea pig and human smooth muscles (4, 20). In contrast, it was reported that in the canine basilar artery, 20-HETE potentiates stretch-induced contraction via PKC-{alpha}-mediated inhibition of a BKCa channel (22). The role of various ion channels in acute and chronic hypoxia in pulmonary vasculature has already been described (32). However, concomitant recordings of BKCa channel activation in HPA smooth muscle cells have not been achieved yet. Nevertheless, it is of physiological interest to realize that 20-HETE displays relaxing effects on both HPA (2, 14) and distal human bronchi (20).

One way to minimize the contribution of surface membrane K+ conductance and to induce a steady-state tension is to shift the equilibrium potential to this monovalent cation using a modified high K+ (80 mM) physiological solution; this maneuver will depolarize the VSM cell. Consequently, the putative activation of the BKCa channel will not be an issue since the cells will remain depolarized as previously reported (20). Under these experimental conditions, 20-HETE displays consistent and concentration-dependent relaxing effects as reported in Fig. 1, which could not be due to membrane hyperpolarization. This unexpected result attested that 20-HETE was likely modifying intracellular processes opposing the tonic effects of the well-characterized Ca2+ entry, which follows KCl depolarization. This observation, derived from a set of control experiments, prompted us to assess the effects of the eicosanoid on the Ca2+ sensitivity of VSM cells.

20-HETE reduces Ca2+ sensitivity of VSM cells. The inherent Ca2+ sensitivity of the MLCK, resulting in MLC phosphatase (MLCP) and contraction and subsequent dephosphorylation by MLCP, is an important mechanism in the regulation of VSM tone (17, 30). Modulation of this mechanism by 20-HETE would explain its overall effects on HPAs. The present data demonstrate that in HPAs, 20-HETE significantly reduces Ca2+ sensitivity in permeabilized preparations. Several studies have suggested that Ca2+-sensitizing mechanisms may also be primed under pathophysiological conditions and especially in PAH (6, 29, 31). It was, therefore, of potential clinical interest to find a lipid mediator that would be able to significantly shift the Ca2+ activation curve toward higher concentrations. In a recent publication, we reported that another eicosanoid, 14,15-EET, induces a reduction in Ca2+ sensitivity in fresh or hyperreactive human bronchi, suggesting that this eicosanoid modulates enzymatic systems such as Rho-Rho kinase and/or PKC/CPI-17 pathways (19).

We were also able to show that 20-HETE pretreatment decreases Ca2+ sensitization induced by PDBu (a PKC activator). Based on evidence provided in the literature, CPI-17 was a likely candidate for PKC phosphorylation involved in modulating myofilament Ca2+ sensitivity (3, 7, 30). Western blot analysis performed on HPA homogenates attests that 20-HETE pretreatments decrease CPI-17 phosphorylation levels, whereas PDBu has the opposite effect. Moreover, 20-HETE pretreatments of HPA increase the expression of p116Rip, as attested by Western blot analysis. This result supports the view that 20-HETE downregulates the Rho kinase-dependent pathway (17). In contrast, it was reported that in coronary arteries, 20-HETE increases the Ca2+ sensitivity and induced contractions, which are dependent on Rho kinase activation (27). Together, these results suggest that eicosanoids, such as 20-HETE, may modulate HPA tone through a shift in intracellular protein regulation, although modifications in gene expression and mRNA transcript cannot be ruled out.

In conclusion, the present study demonstrates that 20-HETE is a potent vascular relaxant in HPAs. This relaxing effect appears mainly related to rapid and long-lasting actions on the Ca2+-dependent processes, which include the activation of K+-selective channels from the surface membrane and the reduced phosphorylation of intracellular regulatory proteins. The activation of the former would facilitate membrane hyperpolarization and reduce Ca2+ entry and tone, whereas the latter would directly decrease the contractility of VSM cells. Thus this study provides new information for the cellular basis of the mechanism of action of 20-HETE and would lay the background for clinical trials toward an improved management of human PAH.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a Canadian Institute for Health Research (CIHR) Grant MOP-57677. C. Guibert was supported by a CIHR/Institut National de la Santé et de la Recherche Médicale fellowship exchange program. C. Morin is a recipient of a doctorate studentship supported by National Sciences and Engineering Research Council. E. Rousseau is a member of the Respiratory Health Network of the Fonds de la Recherche en Santé de Québec (http://rsr.chus.qc.ca).


   ACKNOWLEDGMENTS
 
We thank Dr. John R. Falck for the gift of DDMS compound and Dr. M. Ikebe for the p116Rip antibody.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Rousseau, Le Bilarium, Dept. of Physiology and Biophysics, Université de Sherbrooke, 3001 12th Ave. N, Sherbrooke, J1H 5N4, QC, Canada (e-mail: Eric.Rousseau{at}USherbrooke.ca)

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
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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