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J Appl Physiol 99: 1391-1396, 2005. First published June 16, 2005; doi:10.1152/japplphysiol.00473.2005
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5,6-EET-induced contraction of intralobar pulmonary arteries depends on the activation of Rho-kinase

Jennifer L. Losapio, Randy S. Sprague, Andrew J. Lonigro, and Alan H. Stephenson

Department of Pharmacological and Physiological Science, Saint Louis University, St. Louis, Missouri

Submitted 25 April 2005 ; accepted in final form 11 June 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The mechanism mediating epoxyeicosatrienoic acid (EET)-induced contraction of intralobar pulmonary arteries (PA) is currently unknown. EET-induced contraction of PA has been reported to require intact endothelium and activation of the thromboxane/endoperoxide (TP) receptor. Because TP receptor occupation with the thromboxane mimetic U-46619 contracts pulmonary artery via Rho-kinase activation, we examined the hypothesis that 5,6-EET-induced contraction of intralobar rabbit pulmonary arteries is mediated by a Rho-kinase-dependent signaling pathway. In isolated rings of second-order intralobar PA (1–2 mm OD) at basal tension, 5,6-EET (0.3–10 µM) induced increases in active tension that were inhibited by Y-27632 (1 µM) and HA-1077 (10 µM), selective inhibitors of Rho-kinase activity. In PA in which smooth muscle intracellular Ca2+ concentration ([Ca2+]i) was increased with KCl (25 mM) to produce a submaximal contraction, 5,6-EET (1 µM) induced a contraction that was 7.0 ± 1.6 times greater than without KCl. 5,6-EET (10 µM) also contracted {beta}-escin permeabilized PA in which [Ca2+]i was clamped at a concentration resulting in a submaximal contraction. Y-27632 inhibited the 5,6-EET-induced contraction in permeabilized PA. 5,6-EET (10 µM) increased phosphorylation of myosin light chain (MLC), increasing the ratio of phosphorylated MLC/total MLC from 0.10 ± 0.03 to 0.30 ± 0.02. Y-27632 prevented this increase in MLC phosphorylation. These data suggest that 5,6-EET induces contraction in intralobar PA by increasing Rho-kinase activity, phosphorylating MLC, and increasing the Ca2+ sensitivity of the contractile apparatus.

cytochrome P-450; lung; isolated vessels; Y-27632; U-46619; myosin light chain phosphatase

EPOXYEICOSATRIENOIC ACIDS (EETs) are arachidonic acid metabolites of cytochrome P-450 (CYP450) epoxygenase activity that have been demonstrated to be vasoactive in both the systemic and pulmonary circulations (27). In the systemic vasculature, EETs are most commonly described as vasodilators. The mechanism of EET-induced vasodilation has been studied extensively and depends on hyperpolarization of vascular smooth muscle cells (2, 4, 8, 10) resulting from EET-induced opening of smooth muscle cell potassium channels (2, 4, 7–9, 12, 23, 42). Although EETs also reduce tension in nonresistance extralobar pulmonary artery (PA) segments (30, 32, 33), in small-diameter intralobar PA segments of rabbits and rats (33, 37, 41) all four EET regioisomers (5,6-, 8,9-, 11,12-, and 14,15-EET) were observed to be vasoconstrictors. Because EETs also increase vascular resistance in isolated perfused rabbit lungs (33), the predominant activity of EETs in the lung appears to be vasoconstriction. These data are supported by the observation that inhibition of epoxygenase activity with a selective pharmacological inhibitor attenuated PA contractions to phenylephrine (40), suggesting that endogenous EETs participate in {alpha}-adrenergic contraction of the pulmonary vasculature.

Zeldin et al. (38) reported the most abundant EET regioisomer formed in the rabbit lung was 5,6-EET. Of the four EET regioisomers, 5,6-EET is also the most potent constrictor of rabbit pulmonary arteries (41). The 5,6-EET-induced contraction of rabbit PA requires intact endothelium, cyclooxygenase activity, and activation of the thromboxane/endoperoxide (TP) receptor (33, 41). Therefore, 5,6-EET does not activate the vascular smooth muscle TP receptor directly. It has been proposed that 5,6-EET is either metabolized to a thromboxane-like compound by endothelial cyclooxygenase (COX) or it stimulates the endogenous synthesis of a thromboxane-like compound in the endothelium (33, 41). Previously, we reported that a TP receptor antagonist, ONO-3708, but not a thromboxane synthase inhibitor, OKY-046, inhibited 5,6-EET-induced vasoconstriction in isolated rabbit lungs (33). Therefore, a TP receptor agonist other than thromboxane appears to have mediated the 5,6-EET-induced contraction. However, other than these limited observations, specific features of the signaling mechanism responsible for 5,6-EET-induced PA contraction are unknown.

Contraction of vascular smooth muscle occurs via two related mechanisms. In the first, a rise in cytosolic calcium concentration ([Ca2+]i) results in the formation of a calcium/calmodulin complex that activates myosin light chain kinase (MLCK). Activated MLCK phosphorylates the 20-kDa myosin light chain (MLC) (17), resulting in smooth muscle cell contraction. However, an additional mechanism has been described that is independent of changes in [Ca2+]i but requires activation of Rho-kinase (31) to mediate contraction of vascular smooth muscle. This mechanism is often referred to as calcium sensitization. Calcium sensitization occurs when an agonist that stimulates the activation of Rho-kinase results in inhibition of MLC phosphatase (MLCP) (31). Rho-kinase inhibits MLCP activity by phosphorylation of the myosin-binding subunit of MLCP or by phosphorylation and activation of myosin phosphatase inhibitor protein (CPI-17) (19). While phosphorylated, MLCP is unable to dephosphorylate the MLCK-phosphorylated MLC (P-MLC). The increased P-MLC resulting from reduced MLCP activity results in vascular smooth muscle contraction at a fixed concentration of [Ca2+]i (31).

Recently there has been significant interest regarding the participation of Rho-kinase in the regulation of pulmonary smooth muscle tone (34), especially as it relates to hypoxic pulmonary vasoconstriction (5, 24, 26, 39) and primary pulmonary hypertension (1). There is evidence that Rho-kinase can be activated by hypoxia in PA smooth muscle cells (36) resulting in pulmonary vasoconstriction. The signaling pathway resulting in activation of Rho-kinase can also be initiated through the activation of the TP receptor (11), thus making Rho-kinase a potential participant in 5,6-EET-induced pulmonary vasoconstriction. The aim of the present study was therefore to test the hypothesis that activation of Rho-kinase is required for 5,6-EET-induced increases in active tension in rabbit pulmonary arteries.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animal preparation.   Adult New Zealand White rabbits (2.4–3.0 kg) were anesthetized with pentobarbital sodium (15 mg/kg iv) 10 min after intramuscular administration of ketamine (8 mg/kg) and xylazine (2 mg/kg). A tracheostomy was performed, and a tracheal cannula was inserted. The animals were ventilated via a fixed volume ventilator (Harvard) with room air (tidal volume: 8–10 ml/kg at 15 cycles/min). A catheter was inserted into a carotid artery for administration of heparin (1,000 units iv) 10 min before exsanguination of the animal. After exsanguination, the lungs were removed for isolation of the pulmonary vessels. The Saint Louis University Institutional Animal Care and Use Committee approved the protocols for animal use.

Isolated PA segment protocol.   Intralobar, second-order PA were obtained as described previously (33). Briefly, intralobar PA (1–2 mm OD) were dissected free of extravascular tissue, cut into rings 3–4 mm in length, and suspended in water-jacketed tissue chambers containing 10 ml of a physiological salt solution (PSS) composed of (in mM) 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 0.026 Na-EDTA, and 11.1 glucose. The PSS was gassed with 95% O2-5% CO2 (pH: 7.4) and maintained at 37°C. Each ring was mounted between two stainless steel support wires. Ring tension was measured from one of the support wires attached to an isometric force transducer (FT03, Grass) and recorded continuously on a polygraph (Grass). Each ring was placed under a basal (passive) tension (0.75–1.5 g) determined to result in a maximal contractile response to KCl (60 mM). In some studies, basal tension was increased with 25 mM KCl to achieve a submaximal increase in active tension. Selective epoxidation of arachidonic acid to 5,6-EET was achieved using the method of Corey et al. (3) as described previously (32). 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. At basal tension, concentrations of 5,6-EET (1x10–8–1x10–5 M, delivered in 1 µl of absolute ethanol) were added individually or cumulatively to the rings in 10 ml PSS. Total ethanol concentration in the tissue chambers never exceeded 0.1%. 5,6-EET was added in the presence or absence of selective Rho-kinase inhibitors HA-1077 (10 µM, Sigma, St. Louis, MO) (29) or Y-27632 (1 µM, Calbiochem, La Jolla, CA) (35), each administered in 1 µl H2O. At the concentrations used, the ethanol vehicle for added agents did not alter the basal tension or active tension of the PA rings.

Permeabilization of intralobar PA rings.   PA rings were permeabilized using {beta}-escin to clamp the cytosolic calcium at a fixed concentration (25). PA rings, prepared as described above, were permeabilized at room temperature for 30 min with {beta}-escin (50 µM, Sigma) in PIPES buffer containing (in mM) 10 PIPES, 10 creatine phosphate, 5.1 MgCl2, 87 KCl, 5.2 MgATP, 2 EGTA, 0.001 leupeptin, and 0.001 A-23187 at pH 7.4. The {beta}-escin-containing solution was then replaced with PIPES buffer without {beta}-escin at 37°C and gassed with 95% O2-5% CO2. After basal tension was reestablished, CaCl2 was added until a submaximal increase in active tension (0.2 ± 0.09 g) was measured. Permeabilization was considered to be successful if KCl (60 mM) no longer increased active tension in PA rings. 5,6-EET (10 µM) was added to the permeabilized PA rings in the presence of GTP (20 µM), a concentration that did not alter vessel tension. A concentration of 5,6-EET (10 µM) was chosen for studies in permeabilized PA rings because lower concentrations resulted in less reproducible increases in active tension.

MLC phosphorylation assay.   PA rings, incubated in organ chambers as described above, were exposed to either U-46619 (0.1 µM), a thromboxane mimetic previously reported to stimulate Rho-kinase activity, or 5,6-EET (10 µM) in the presence or absence of the Rho-kinase inhibitor Y-27632 (1 µM, 30 min preincubation). As the vessels used in these studies exhibit some basal tension in organ chambers, there is a low but measurable level of MLC phosphorylation present under basal conditions that may result from calcium-driven MLCK activity or a basal level of Rho-kinase activation. To maximize the differences between basal MLC phosphorylation, 5,6-EET-induced MLC phosphorylation, and 5,6-EET-induced MLC phosphorylation inhibited by the Rho-kinase inhibitor Y-27632, 5,6-EET was administered at 10 µM, a concentration that resulted in pulmonary artery contraction about twofold greater than that resulting from 1 µM 5,6-EET. At the maximum active tension recorded, the organ chamber was lowered and the PA ring was immediately immersed in a solution containing acetone, DTT (10 mM), and TCA (10%) chilled on dry ice. After a 30-min incubation in this solution, the PA ring was washed three times with acetone-DTT and air-dried. When dry, the tissue was dissolved in 150 µl 9 M urea loading buffer containing (in mM), 20 Tris base, 23 glycine, and 10 DTT.

Western immunoblot analysis.   For Western immunoblots, samples were loaded onto acrylamide gels containing 40% glycerol, 10% acrylamide, 0.5% bis-acrylamide, 20 mM Tris, and 23 mM glycine. Proteins were resolved by electrophoresis at 450 V for 2 h and transferred to nitrocellulose membranes (100 V, 60 min, 4°C). The membranes were blocked for 1 h in a Tris-buffered saline (TBS) solution containing 10 mM sodium phosphate, 150 mM NaCl, and 5% nonfat milk at pH 7.4. After blocking, membranes were incubated overnight in TBS containing a 1:1,000 dilution of a rabbit polyclonal antibody directed against MLC (Santa Cruz Biotechnology, Santa Cruz, CA). After washing with TBS containing Tween-20 (0.1%), the membranes were incubated with a 1:2,000 dilution of an anti-rabbit secondary antibody conjugated with horseradish peroxidase (Amersham) for 1 h at room temperature. Proteins were detected with enhanced chemiluminescence (Amersham Biosciences). Phosphorylation of MLC results in a mobility shift of the P-MLC (39). Phosphorylated (lower band) and unphosphorylated (upper band) MLC were measured by densitometry. Phosphorylation of MLC was expressed as the ratio of P-MLC to total (phosphorylated plus unphosphorylated) MLC.

Statistical methods.   All values are expressed as means ± SE. Differences between experimental groups were determined by ANOVA. If the F-ratio indicated significant differences, a least significant difference test was used to establish differences between individual sample means. When appropriate, a Student's t-test for paired data was used. Values of P < 0.05 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Effect of Rho-kinase inhibition with Y-27632 (1 µM) on 5,6-EET-induced increases in active tension.   In isolated, second-order intralobar PA segments, administration of 5,6-EET increased active tension in a concentration-dependent manner (Fig. 1) as had been reported previously (33). Preincubation for 30 min with the Rho-kinase inhibitor Y-27632 (1 µM) significantly inhibited this increase in tension (Fig. 1). A chemically dissimilar inhibitor of Rho-kinase, HA-1077 (10 µM), inhibited 5,6-EET-induced increases in active tension in a manner similar to Y-27632 (Fig. 1).



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  		Fig. 1. Effect of a 30-min preincubation with Rho-kinase inhibitors Y-27632 (1 µM) and HA-1077 (10 µM) on cumulative increases in active tension induced by 5,6-epoxyeicosatrienoic acid (EET) in isolated rabbit pulmonary artery (PA) rings. *P < 0.05 from baseline tension, {dagger}P < 0.05 from vehicle treated PA rings. Values are means ± SE; n = 7.

 
If 5,6-EET-induced contraction of PA rings is mediated by increased Ca2+ sensitivity, then the 5,6-EET-induced increase in PA active tension should be augmented by increasing [Ca2+]i (25). As shown in Fig. 2, the increase in active tension resulting from 5,6-EET (1 µM) was 7.0 ± 1.6 times greater when basal tension in the PA rings was increased to ~20% of the maximal KCl-induced tension by opening vascular smooth muscle voltage-gated Ca2+ channels with 25 mM KCl (Fig. 2).



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  		Fig. 2. Typical recording of the contraction evoked by 5,6-EET (1 µM) in the presence (A) and the absence (B) of a submaximal depolarizing contraction resulting from incubation of the PA ring with KCl (25 mM). C: effect of KCl (25 mM) on 5,6-EET-induced active tension in PA rings. *P < 0.01 from vehicle + 5,6-EET. Values are means ± SE; n = 5.

 
Effect of Rho-kinase inhibition with Y-27632 (1 µM) on KCl (60 mM)-induced increases in active tension.   To demonstrate that Y-27632 is selective for agonists stimulating PA contraction via a Rho-kinase-dependent pathway, KCl, a calcium/ calmodulin-dependent contractile agonist, was added to the vessels after a 30-min incubation with Y-27632 (1 µM). A concentration of 1 µM Y-27632 was chosen for inhibition of Rho-kinase activity in these studies because previously it was determined that Y-27632 at concentrations of up to 3 µM did not affect KCl-induced contractions in rat PA rings (13). Incubation of rabbit PA rings with Y-27632 (1 µM), a concentration that attenuated 5,6-EET-induced increases in PA contraction by 85.4 ± 5.3% at 10 µM 5,6-EET (Fig. 1), did not inhibit PA contraction resulting from incubation with 60 mM KCl (Fig. 3).



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  		Fig. 3. Effect of a 30-min preincubation with Y-27632 (1 µM) on the increase in active tension induced by KCl (60 mM) in isolated rabbit PA rings. Values are means ± SE; n = 4.

 
Effect of 5,6-EET and U-46619 on MLC phosphorylation in second-order, intralobar rabbit pulmonary vessels.   Administration of U-46619 (0.1 µM), a TP receptor agonist reported previously to increase MLC phosphorylation in rat pulmonary vasculature (20), resulted in a Y-27632-sensitive increase in the ratio of P-MLC to total MLC in rabbit PA (Fig. 4A) determined by an electrophoretic mobility shift of P-MLC. U-46619-induced phosphorylation of rabbit PA MLC was used as a positive control in tissue from each rabbit in which 5,6-EET-induced MLC phosphorylation was examined. 5,6-EET increased the ratio of P-MLC/total MLC from a control value of 0.10 ± 0.03 to 0.30 ± 0.02 (Fig. 4B, P < 0.01, n = 7). Incubation of the PA rings with Y-27632 (1 µM) for 30 min before adding the 5,6-EET resulted in a 49.4 ± 10.4% reduction in MLC phosphorylation. These results are consistent with the hypothesis that 5,6-EET-induced phosphorylation of MLC is Rho-kinase dependent.



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  		Fig. 4. Effect of 5,6-EET on the phosphorylation of myosin light chain (MLC) in rabbit PA rings. A: representative Western blot showing a shift in the mobility of MLC in second-order intralobar PA rings with 5,6-EET (10 µM) and U-46619 (0.1 µM) in the presence and absence of Y-27632 (1 µM) preincubated with the PA rings for 30 min. MLC and P-MLC refer to the nonphosphorylated and phosphorylated forms of MLC, respectively. B: ratio of P-MLC to total MLC in response to 5,6-EET (10 µM) incubation in the absence and presence of Y-27632 (1 µM) preincubated with the PA rings for 30 min. *P < 0.01 from 5,6-EET/Y-27632 (–/–). {dagger}P < 0.01 from 5,6-EET/Y-27632 (+/–). Values are means ± SE; n = 7.

 
Effect of 5,6-EET on calcium sensitization in permeabilized second-order, intralobar rabbit pulmonary vessels.   In the presence of a submaximal CaCl2-induced increase in active tension (0.20 ± 0.09 g), administration of 5,6-EET (10 µM) to permeabilized PA rings increased active tension by 0.52 ± 0.11 g (Fig. 5). This 5,6-EET-induced contractile response was decreased by 88.9 ± 8.2% after preincubation of the vessels for 30 min with Y-27632 (1 µM).



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  		Fig. 5. Effect of 5,6-EET on {beta}-escin-permeabilized PA rings. Typical trace showing the increase in active tension resulting from administration of 5,6-EET (10 µM) to {beta}-escin-permeabilized second-order intralobar PA rings in the presence of a submaximal CaCl2-induced contraction in the absence (A) and the presence (B) of Y-27632 (1 µM) preincubated with the PA rings for 30 min. C: summary of the effect of 5,6-EET on {beta}-escin-permeabilized PA rings. *P < 0.01 from baseline tension. {dagger}P < 0.01 from 5,6-EET/Y-27632 (+/–). Values are means ± SE; n = 6.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
In the present investigation, we provide the first evidence that 5,6-EET increases active tension in isolated PA rings through a mechanism that depends on Rho-kinase activation. These results suggest that 5,6-EET induces contraction in intralobar pulmonary arteries by increasing the Ca2+ sensitivity of the contractile apparatus. Similar inhibition of 5,6-EET-induced contraction of PA was obtained with two chemically dissimilar selective Rho-kinase inhibitors, Y-27632 (35) and HA-1077 (29). Although KCl (60 mM) also increased active tension in these isolated PA rings, the KCl-induced contraction was not inhibited by Y-27632 at a concentration that largely inhibited EET-induced PA contraction (Fig. 1), indicating that the response to KCl in rabbit PA is independent of Rho-kinase activation. KCl-induced contraction of vascular smooth muscle is mediated by smooth muscle cell membrane depolarization leading to voltage-dependent Ca2+ influx (18). Although there is some disagreement in the literature regarding the participation of Rho-kinase in vascular smooth muscle contractions induced by depolarizing concentrations of KCl, we show here, as others have reported previously (13, 25), that when administered at 1 µM, Y-27632 inhibits Rho-kinase activity but does not inhibit KCl-induced contractions of intralobar rabbit PA. In isolated rat pulmonary arteries, Hyvelin et al. (13) reported that Y-27632 inhibited Rho-kinase-mediated vascular smooth muscle contraction at 1 µM but did not inhibit KCl-induced contraction at concentrations up to 3 µM; however, at 10 µM, Y-27632 did attenuate KCl-induced contractions. Others, including Roberson et al. (26), Fagan et al. (5), and Janssen et al. (16), reported that Y-27632 inhibited hypoxic pulmonary vasoconstriction and KCl-induced pulmonary vasoconstriction in rats, mice, and pigs, respectively. In each of these studies, Y-27632 inhibited KCl-induced contraction at the higher 10 µM concentration found to be nonselective by Hyvelin et al. (13). However, in isolated rabbit aorta (28) and in rat caudal artery (22), in addition to demonstrating that Y-27632 inhibited KCl-induced contractions, KCl was reported to increase the amount of an active GTP-bound form of RhoA and to increase P-MLC, consistent with Rho-kinase-dependent Ca2+ sensitization. Therefore, it is possible that KCl-dependent activation of Rho-kinase occurs in a species- and/or tissue-dependent manner.

Additional support for the hypothesis that 5,6-EET-induced contraction of intralobar rabbit PA is mediated by activation of Rho-kinase comes from the studies in which the contraction of PA rings to 5,6-EET was potentiated in the presence of a submaximal contraction resulting from preincubation with 25 mM KCl, as has been reported for Rho-kinase-mediated contraction of porcine coronary arteries with 20-HETE (25), another CYP450 metabolite of arachidonic acid. The mechanism of this potentiation depends on the increased vascular smooth muscle [Ca2+]i resulting from KCl-induced Ca2+ influx through voltage-operated Ca2+ channels. The increased [Ca2+]i can activate MLCK, which in turn increases the rate of MLC phosphorylation, resulting in contraction. 5,6-EET-induced Rho-kinase activation in the presence of enhanced MLCK activity would therefore result in a greater 5,6-EET-induced contraction as demonstrated in Fig. 3.

To demonstrate that 5,6-EET increased MLC phosphorylation in rabbit PA rings through a Rho-kinase-dependent mechanism, we measured increased P-MLC by observing a mobility shift in Western immunoblots (39). In these studies, we used U-46619 as a positive control that increases vascular smooth muscle contraction through a mechanism relying almost exclusively on the activation of Rho-kinase and not as a result of an increase in [Ca2+]i (15). Incubation of intralobar rabbit PA rings with either U-46619 or 5,6-EET resulted in an increase in Y-27632-inhibitable MLC phosphorylation, suggesting that the increased P-MLC depended on activation of Rho-kinase and increased Ca2+ sensitization rather than an increase in [Ca2+]i.

Although 5,6-EET has been reported to increase [Ca2+]i in vascular smooth muscle cells in porcine coronary vessels (6), 5,6-EET does not contract pulmonary vessels after removal of the endothelium (33, 41), suggesting that 5,6-EET does not contract PA directly by increasing smooth muscle cell [Ca2+]i. However, to confirm that 5,6-EET-induced contraction of intralobar rabbit PA is truly independent of increased [Ca2+]i, 5,6-EET-induced contraction was studied in intralobar PA rings permeabilized with {beta}-escin. In {beta}-escin-permeabilized vascular rings, the [Ca2+]i is clamped at a concentration resulting in the maintenance of a submaximal contraction. In these permeabilized PA rings, 5,6-EET increased active tension. As with intact vessels, the 5,6-EET-induced contraction in these {beta}-escin-permeabilized PA rings was inhibited by Y-27632, demonstrating that Rho kinase mediated increased Ca2+ sensitization.

Although this is the first report describing the dependence of 5,6-EET-induced vascular contraction on Rho-kinase activation, vascular contraction induced by other eicosanoids, e.g., 20-HETE in porcine coronary artery (25), thromboxane A2 in dog (15) and rat (20) pulmonary vasculature, PGF2{alpha} in rabbit aorta (14), and PGE2 in guinea pig aorta has been reported to require Rho-kinase activation. In addition, in airway smooth muscle, 5-oxo-6,8,11,14-eicosatetraenoic acid, a lipoxygenase metabolite of arachidonic acid was reported to contract airway smooth muscle by a Rho-kinase-dependent mechanism (21).

In summary, we showed that in intralobar rings of rabbit PA, 5,6-EET, a CYP450 metabolite of arachidonic acid, induces a Rho-kinase-dependent increase in active tension. 5,6-EET is a major metabolite of arachidonic acid in the rabbit pulmonary vasculature and therefore may contribute to pulmonary vascular contraction resulting from conditions in which endogenous arachidonic acid is liberated by an increase in phospholipase activity.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grants HL-52675 (A. H. Stephenson) and HL-51298 and HL-64180 (R. S. Sprague) and a predoctoral grant from the American Heart Association (0415364Z, J. L. Losapio).


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors thank Jo Schreiweis and Elizabeth Bowles for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. H. Stephenson, Dept. of Pharmacological and Physiological Science, St. Louis, MO 63104 (e-mail: stephens{at}slu.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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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