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Cardiovascular Research 2005 65(2):487-494; doi:10.1016/j.cardiores.2004.10.029
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Copyright © 2004, European Society of Cardiology

The synthesis of 20-HETE in small porcine coronary arteries antagonizes EDHF-mediated relaxation

Voahanginirina Randriamboavonjya, Ladislau Kissb, John R. Falckc, Rudi Bussea and Ingrid Fleminga,*

aVascular Signalling Group, Institut für Kardiovaskuläre Physiologie, Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany
bZentrum der Inneren Medizin, Justus Liebig Universität Gieβen, Klinikstrasse 36, D-35392 Gieβen, Germany
cDepartment of Biochemistry, University of Texas Southwestern Medical Center (J.R.F.), Dallas Texas 75390, USA

* Corresponding author. Tel.: +49 69 6301 6972; fax: +49 69 6301 7668. Email address: fleming{at}em.uni-frankfurt.de

Received 13 July 2004; revised 11 October 2004; accepted 21 October 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Exogenous application of 20-hydroxyeicosatetraenoic acid (20-HETE) to small (300–500 µm) porcine coronary arteries elicits contraction by activating the Rho kinase and increasing the sensitivity of contractile proteins to Ca2+. Here, we determined whether 20-HETE is involved in the regulation of coronary artery tone as well as its role in the modulation of endothelium-derived hyperpolarizing factor (EDHF)-mediated responses.

Methods and results: Small porcine coronary arteries expressed cytochrome P450 (CYP) 4A, as demonstrated by Western blot analysis, and generated 20-HETE. Moreover, 20-HETE production was increased two- and threefold over basal levels in response to isometric stretch or the thromboxane analogue U46619 [GenBank] , respectively, and was inhibited by the CYP 4A inhibitor N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS). In vascular reactivity studies, DDMS attenuated U46619 [GenBank] -induced contractions and induced a concentration-dependent but endothelium-independent relaxation of precontracted arterial rings. Endogenously generated 20-HETE significantly inhibited the EDHF-mediated relaxation of coronary arteries, which was potentiated by the phospholipase A2 inhibitors AACOCF3 and ONO-RS-082, as well as by the {omega}-hydroxylase inhibitors 17-octadecynoic acid and DDMS. EDHF-mediated relaxation was not affected by either the nonselective epoxygenase inhibitors miconazole and clotrimazole or the CYP 2C inhibitor sulfaphenazole but was abolished by the Na-K-ATPase inhibitor, ouabain. Exogenous application of 20-HETE inhibited EDHF-mediated relaxations and caused a concomitant increase in the phosphorylation of protein kinase C{alpha} (PKC{alpha}). This effect was reversed by the PKC inhibitor Ro-318220 and mimicked by the PKC activator phorbol-12 myristate 13-acetate.

Conclusions: These results indicate that vascular tone in small porcine coronary arteries is partly determined by the endogenous production of 20-HETE. In addition, 20-HETE functionally antagonizes EDHF-mediated relaxation via a PKC{alpha}-dependent mechanism, probably involving the inhibition of the Na-K-ATPase.

KEYWORDS Endothelial factors; Na/K-pump; Protein kinase C; Stretch/m-e coupling; Vasoconstriction/dilation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
The regulation of vascular tone involves a complex interplay between vasoconstrictor and vasodilator stimuli. The myogenic response is a main determinant of vascular tone in situ and data gathered over the last 15 years have convincingly shown a link between vascular 20-hydroxyeicosatetraenoic acid (20-HETE) generation and myogenic responses in renal, cerebral, and mesenteric arteries [1–4]. In smooth muscle cells, 20-HETE is endogenously produced by {omega}-hydroxylases of the cytochrome P450 (CYP) 4A family following an increase in [Ca2+]i [5,6]. Once formed, 20-HETE increases smooth muscle tone (and enhances sensitivity to vasoconstrictor agents) by inhibiting large conductance Ca2+-dependent K+ channels (KCa+) [7], thus inducing depolarization and contraction (for review, see Ref. [8]). 20-HETE has also been linked with the activation of protein kinase C (PKC) [9], the inhibition of the Na-K-ATPase [10,11] as well as to the activation of the Rho kinase and the sensitization of the contractile apparatus to Ca2+ [12].

Endothelium-derived vasodilator autacoids such as nitric oxide (NO), prostacyclin and the endothelium-derived hyperpolarizing factor (EDHF) are able to modulate the myogenic response. To date, only NO has been reported to interfere with the formation and actions of 20-HETE. Indeed, the NO-mediated inhibition of 20-HETE formation has been proposed to account for the natriuretic and diuretic actions of NO [13], as well as the cyclic GMP-independent relaxant effects of NO in renal and cerebral arteries [14–16].

Since 20-HETE and EDHF appear to share common targets; that is, both may regulate vascular tone by influencing KCa+ channels as well as the Na-K-ATPase, the aim of the present investigation was to determine whether the tone of small porcine coronary arteries reflects a balance between the activity of the vasodilator EDHF and the endogenous generation of the constrictor 20-HETE.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Materials
AACOCF3 was purchased from Calbiochem, bradykinin and charybdotoxin were from Bachem Biochemica (Heidelberg, Germany), 2-(p-Amylcinnamoyl)amino-4-chlorobenzoic acid (ONO-RS-082) was from Biomol (Hamburg, Germany), U46619 [GenBank] (9,11-dideoxy-epoxymethano-prostaglandin F2) was from Alexis Biochemicals (Grünberg, Germany), and 20-HETE was from Cayman Chemical (Massy, France). N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) was synthesized in the laboratory of Dr J.R. Falck as previously described [17]. Phorbol-12-myristate-13-acetate (PMA) and all other drugs were purchased from Sigma (Deisenhofen, Germany).

2.2. Vascular reactivity studies
Porcine hearts were obtained from a local slaughterhouse, placed immediately into ice-cold HEPES–Tyrode solution (composition in mmol/L: NaCl 137, KCL 2.7, CaCl2 1.8, MgCl2 0.5, NaH2PO4 0.36, glucose 5, HEPES 10), and transported to the laboratory. Second branches of coronary arteries (internal diameter 300–500 µm) were dissected, cleaned of adventitial adipose and connective tissue, and cut into 4-mm-long rings. These rings were mounted on stainless steel triangles connected to a force transducer (Hugo Sachs Elektronik-Harvard Apparatus, Germany) and a rigid support for measurement of isometric force in organ baths containing Tyrode's solution of the following composition (mmol/L): NaCl 132, KCl 4, CaCl2 1.6, MgCl2 0.98, NaHCO3 23.8, NaH2PO4 0.36, glucose 10, Ca-Titriplex 0.05, diclofenac 0.01, and gassed with 20% O2, 5% CO2, and 75% N2 to give a pO2 of approximately 140 mmHg and pH 7.4 at 37 °C, as described [12]. Passive tension was gradually adjusted over a 60-min period to 1 g, thereafter arterial rings were repeatedly exposed to a modified Tyrode's solution rich in KCl (80 mmol/L) until stable contractions were obtained. The presence of functional endothelium was assessed in all preparations by the ability of bradykinin (1 µmol/L) to induce relaxation of vessels precontracted with U46619 [GenBank] (0.1 µmol/L) and vessels exhibiting less than 80% relaxation were discarded.

2.3. Immunoblotting
Endothelium-intact rings of porcine coronary artery were incubated for 30 min at 37 °C in modified Tyrode's solution and stimulated as described in Results. Vessels were then frozen in liquid N2, ground to a powder and suspended in Triton X-100 lysis buffer. Following centrifugation (14,000 x g, 15 min at 4 °C) Triton-soluble and -insoluble proteins were heated with SDS-PAGE sample buffer, separated by SDS-PAGE (8%), and transferred to a nitrocellulose membrane as previously described [18]. Proteins were detected using their respective antibodies, and visualized by enhanced chemiluminescence using a commercially available kit (Amersham, Germany).

The polyclonal CYP 4A antibody was kindly provided by Dr. David Harder (Milwaukee, WI) and the CYP 2C antibody was purified by Eurogentec (Seraing, Belgium) from the serum of rabbits immunized with a CYP 2C9 peptide (65-2-487LPPGPTPLPIC). The phosphoprotein kinase C (PKC pan) and the phospho-PKC{alpha} antibodies were from Cell Signaling Technology (Frankfurt am Main, Germany), the PKC{alpha} antibody was from BD Biosciences Pharmingen (Heidelberg, Germany), and the β-actin antibody from Sigma (Deisenhofen, Germany).

2.4. Measurement of 20-HETE and EET levels in isolated arteries
Coronary artery rings were mounted in organ chambers as described above. Rings were either incubated without stretching or basal tension was adjusted to 1 g for 60 min. Thereafter, vessels were rapidly frozen in liquid nitrogen and ground to a powder. 20-HETE levels were assessed as described [19] with slight modifications. Briefly, following homogenization in 0.2 mol/L KCl containing 0.01% butylated hydroxytoluene (BHT) as an antioxidant and prostaglandin B1 (PGB1) as an internal standard, 20-HETE was extracted from the samples by addition of an equal volume of ethyl acetate containing 0.005% BHT and 0.1% formic acid. This was repeated twice and the combined organic phases were evaporated under a stream of nitrogen. Dried samples were dissolved in acetonitrile/methanol/water (40:10:50, v/v/v) and subjected to isocratic reversed-phase (RP) HPLC with online photodiode array detection (PDAD). RP-HPLC was carried out on octadecylsilyl columns with a mobile phase of acetonitrile/methanol/water/formic acid (52:6:42:0.001). 20-HETE was detected at 204 nm, identified by spectra plot on the peak maximum and co-elution with the commercial 20-HETE standard, and assessed by internal standard method using PGB1. Analysis of full UV spectra (190–340 nm) provided by PDAD allowed accurate identification and quantification of 20-HETE by peak purity control and subtraction of possible coeluting material.

2.5. Statistical analysis
Relaxation is expressed as a percentage of the contraction obtained with U46619 [GenBank] and Rmax represents the maximal relaxation recorded in response to the cumulative addition of a given agonist. pD2 (–log EC50) values were calculated by using commercially available software (Prism 2.01, GraphPAD software, San Diego, CA). Data are expressed as mean ± S.E.M. and statistical evaluation was performed using Students t test for unpaired data or one-way analysis of variance (ANOVA) followed by a Bonferroni's t test, where appropriate. Values of P<0.05 were considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. CYP 4A protein expression and 20-HETE production in small porcine coronary arteries
The expression of CYP 4A and CYP 2C protein was assessed in large and small porcine coronary arteries by Western blotting. The CYP 4A antibody used recognized a 52-kDa band in porcine kidney as well as in the small coronary arteries while only a weak signal was detected in the large porcine coronary artery (Fig. 1A). The CYP 2C epoxygenase, on the other hand was clearly expressed in large porcine coronary arteries while only a weak signal could be detected in smaller arteries.


Figure 1
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  		Fig. 1 CYP expression and 20-HETE production in porcine coronary arteries. (A) Representative immunoblots showing the expression of CYP 4A and CYP 2C isoforms in small (sPCA) and large (lPCA) porcine coronary arteries. A sample of porcine kidney was used as a positive control (pc) for CYP 4A and CYP 2C. The β-actin blot is included to demonstrate equal loading of proteins. Identical results were obtained in three additional experiments. (B) Bar graph showing the effect of isometric stretch (IS, 1 g for 60 min) in the absence and presence of U46619 (IS+U, 1 µmol/L, 10 min) on the production of 20-HETE. Experiments were performed in the absence (CTL) and presence of DDMS (DD, 25 µmol/L) and in the continuous presence of N{omega}-nitro-L-arginine (300 µmol/L) and diclofenac (10 µmol/L). Data represent the mean ± S.E.M. from seven different experiments; **P<0.01 vs. CTL (nonstretched vessels) and §P<0.05, § §P<0.01 versus in the absence of DDMS.

 
A basal production of 20-HETE was detected in small porcine coronary arteries maintained without stretch. DDMS (25 µmol/L) slightly, but nonsignificantly, reduced 20-HETE levels in these arteries (Fig. 1B). Isometric stretch of the vessel (1 g, 60 min), however, led to a 1.6-fold increase in 20-HETE production while isometric stretch and U46619 [GenBank] increased 20-HETE production by 2.1-fold. Both responses were prevented by the CYP 4A inhibitor (Fig. 1B).

3.2. Effect of CYP 4A inhibition on coronary artery tone
To determine whether 20-HETE was generated in small porcine coronary arteries and contributes to the agonist-induced development of vascular tone, we assessed the effects of DDMS using two different protocols.

Preincubation of small porcine coronary artery rings with DDMS (25 µmol/L) resulted in a pronounced rightward shift in the U46619 [GenBank] -induced concentration–contraction curve (Fig. 2A) and slightly reduced the maximal contraction obtained. The pD2 values for U46619 [GenBank] were 7.21 ± 0.13 in the presence of solvent versus 3.29 ± 0.12 in the presence of DDMS (P<0.05, n=6). The maximal contraction to U46619 [GenBank] (1 µmol/L) was 79.7 ± 6.8% in the presence of solvent versus 58.6 ± 12.3% in the presence of DDMS (P<0.05, n=6).


Figure 2
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  		Fig. 2 Effect of CYP 4A inhibition on coronary artery tone. (A) Concentration–response curves showing the effect of DDMS (25 µmol/L) on the U46619-induced contraction. (B) Concentration–relaxation curves showing the effect of solvent (CTL, 0.05% ethanol) or DDMS on the tone of arteries precontracted with U46619. Experiments were performed using endothelium-intact (+E) and endothelium-denuded arteries (–E). All experiments were performed in the presence of diclofenac (10 µmol/L) and the data represent the mean ± S.E.M. from three to five different experiments.

 
In small coronary artery rings precontracted with U46619 [GenBank] to 80% of the maximal KCl (80 mmol/L)-induced contraction, DDMS (5–25 µmol/L) elicited a concentration-dependent but endothelium-independent relaxation (Fig. 2B). DDMS was however unable to induce relaxation in large porcine coronary arteries which express little or no CYP 4A (data not shown).

3.3. Pharmacological characterization of the endothelium-dependent relaxation of small porcine coronary arteries
In U46619 [GenBank] -precontracted coronary artery rings, bradykinin elicited endothelium- and concentration-dependent relaxations. The concentration–relaxation curve to bradykinin was shifted to the right (P<0.05, n=7) and the maximal relaxation slightly but significantly (P<0.05, n=7) attenuated when experiments were performed in the presence of N{omega}-nitro-L-arginine (L-NA, 300 µmol/L, Fig. 3A). This NO- and prostacyclin-independent relaxation can be attributed to an EDHF since it was sensitive to depolarizing concentrations of KCl, as well as to the combination of the KCa+ channel inhibitors charybdotoxin and apamin (each 100 nmol/L). The EDHF-mediated relaxation of small porcine coronary arteries was also sensitive to the Na-K-ATPase inhibitor ouabain (500 nmol/L, Fig. 3A).


Figure 3
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  		Fig. 3 Pharmacological characterization of the endothelium-dependent relaxation of small porcine coronary arteries. Concentration–relaxation curves to bradykinin (Bk) showing the effect of solvent (CTL), N{omega}-nitro-L-arginine (L-NA, 300 µmol/L; A–D) in the absence and in the presence of the combination of apamin and charybdotoxin (CbTx+Apa, each 100 nmol/L; A), ouabain (500 nmol/L; A), AACOCF3 (AAC, 10 µmol/L; B), ONO-RS-082 (ONO, 10 µmol/L; B), clotrimazole (Clotri, 3 µmol/L; C), miconazole (Mico, 3 µmol/L; C), sulfaphenazole (Sulfa, 10 µmol/L; C), DDMS (25 µmol/L; D), or 17-ODYA (10 µmol/L; D). All experiments were performed in the presence of diclofenac (10 µmol/L) and the data represent the mean ± S.E.M. from six different experiments.

 
Since the EDHF-mediated relaxation of larger porcine coronary arteries has been linked to the activation of phospholipase A2 (PLA2) and the generation of epoxyeicosatrienoic acids (EETs) by a CYP epoxygenase [20], we determined the effects of interfering with these processes on the relaxation of small coronary arteries.

The PLA2 inhibitors, AACOCF3 (10 µmol/L) and ONO-RS-082 (10 µmol/L) significantly enhanced the EDHF-mediated relaxation to bradykinin (Fig. 3B). pD2 values were: 6.89 ± 0.41 vs. 7.32 ± 0.117 and 7.4 ± 0.14 for control, AACOCF3, and ONO-RS-082, respectively (n=6, P<0.05). Inhibition of CYP epoxygenases using miconazole (3 µmol/L), clotrimazole (3 µmol/L), or sulfaphenazole (1 µmol/L) failed to influence the bradykinin-induced EDHF-mediated relaxation (Fig. 3C). However, the {omega}-hydroxylase inhibitors 17-octadecynoic acid (17-ODYA, 10 µmol/L) and DDMS (25 µmol/L) induced a significant leftward shift in the concentration–relaxation curve to bradykinin (Fig. 3D). pD2 values were 6.85 ± 0.20 vs. 7.42 ± 0.06 and 7.50 ± 0.10 for control, DDMS, and 17-ODYA, respectively (n=6, P<0.01).

3.4. Effect of 20-HETE on EDHF-mediated relaxation
Since the EDHF-mediated relaxation of small porcine coronary arteries was sensitive to ouabain and 20-HETE is reported to inhibit the activation of the Na-K-ATPase via a protein kinase C-dependent process [11], we assessed the effect of 20-HETE on EDHF-mediated relaxation.

Incubation of precontracted rings of small coronary artery with 20-HETE (1 µmol/L) significantly inhibited EDHF-mediated relaxations (Fig. 4A). Rmax was reduced from 79.4 ± 7.3% in the presence of solvent to 36.6 ± 15.7% in the presence of 20-HETE (P<0.01, n=6). In arteries pretreated with 20-HETE in the presence of the PKC inhibitor Ro 31-8220 (300 nmol/L), Rmax was restored to 86.4 ± 6.9% (P<0.01, n=6; Fig. 4A). EDHF-mediated relaxations were also significantly inhibited by the PKC activator PMA (1 µmol/L), an effect that was also sensitive to Ro 31-8220. Rmax was reduced from 76.8 ± 4.3% in the presence of solvent to 6.3 ± 3.2% in the presence of PMA but increased to 82.5 ± 7.5% in the presence of PMA and Ro 31-8220 (P<0.001, n=5; Fig. 4B).


Figure 4
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  		Fig. 4 Effect of exogenous 20-HETE and PMA on the EDHF-mediated relaxation of small porcine coronary arteries. Concentration–relaxation curves to bradykinin (Bk) obtained in the presence of (A) 20-HETE and (B) PMA (1 µmol/L). Experiments were performed in the absence and presence of Ro-318220 (Ro, 300 nmol/L) and in the continuous presence of N{omega}-nitro-L-arginine (300 µmol/L) and diclofenac (10 µmol/L). The data represent the mean ± S.E.M. from six different experiments.

 
3.5. Effect of 20-HETE on PKC activation
Since the inhibitory effect of 20-HETE on EDHF-mediated relaxations was sensitive to a PKC inhibitor, we assessed the effect of 20-HETE on the phosphorylation of PKC in the Triton X-100-insoluble fraction of small porcine coronary arteries which is thought to contain active PKC isoforms [21]. The phospho-PKC antibody used detects PKC{alpha}, βI, βII, {zeta}, {varepsilon}, and {delta} isoforms only when phosphorylated at a carboxy terminal residue.

A basal PKC phosphorylation was detected in the Triton X-100-insoluble fraction of unstimulated arteries. PMA and 20-HETE increased PKC phosphorylation, effects that were prevented by pretreatment with Ro 31-8220. Moreover, isometric stretch, which increases 20-HETE production in small coronary arteries, also increased PKC phosphorylation. The latter response was prevented by the PKC inhibitor as well as by DDMS (Fig. 5A).


Figure 5
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  		Fig. 5 Effect of PMA, 20-HETE and isometric stretch on the phosphorylation of PKC in small porcine coronary arteries. (A) The representative Western blots show the effect of PMA (1 µmol/L, 10 min), 20-HETE (1 µmol/L, 10 min), and isometric stretch (1 g, 60 min) on the phosphorylation of PKC (p-PKC) in the Triton X-100-insoluble (Tx-insol) fraction of arterial homogenates. Experiments were performed in the presence of either solvent (S, 0.05% ethanol), Ro-318220 (Ro, 300 nmol/L), or DDMS (DD, 25 µmol/L). The bar graph summarizes data obtained in 6 separate experiments; **P<0.01, ***P<0.01 vs. control (CTL). (B) Representative Western blots showing the effect of PMA, 20-HETE, and isometric stretch on the phosphorylation of PKC{alpha} (p-PKC{alpha}) in the Triton X-100-insoluble fraction of arterial homogenates. Similar results were obtained in two additional experiments.

 
As PKC{alpha} has been implicated in 20-HETE-mediated responses [22,23], experiments were repeated using an antibody that specifically recognizes the phosphorylated form of PKC{alpha}. We observed that PMA, 20-HETE, and isometric stretch induced the Ro 31-8220-sensitive phosphorylation of PKC{alpha} in the cell membrane (Triton X-100-insoluble) fraction. Slight increases in PKC{alpha} levels in the membrane fraction were only observed in vessels treated with PMA. Most importantly, the isometric stretch-induced phosphorylation of PKC{alpha} in the membrane fraction was sensitive to DDMS.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The results of the present study indicate that small branches of porcine coronary arteries express the CYP 4A {omega}-hydroxylase and generate 20-HETE when subjected to isometric stretch or stimulated with the thromboxane analogue U46619. [GenBank] The 20-HETE endogenously generated in this manner functionally antagonizes the EDHF-mediated relaxation of these vessels. At least part of the action of 20-HETE can be attributed to the activation of PKC which inactivates the Na-K-ATPase, the main effector of EDHF-mediated relaxation in these vessels.

Several distinct cellular mechanisms have been proposed to account for the phenomenon of EDHF-mediated relaxation (for a recent review, see Ref. [24]). To date, the activation of KCa+ channels in endothelial cells and the subsequent efflux of K+ and activation of inwardly rectifying K+ channels or the Na-K-ATPase in smooth muscle cells have been suggested to account for relaxation in various vascular beds [25]. In coronary arteries, two additional "types" of EDHF have been proposed: the generation of hydrogen peroxide (H2O2) [26] and the generation of EETs from arachidonic acid by CYP epoxygenases [20,27]. Although the role for H2O2 in EDHF-mediated vasodilatation is highly controversial, the "CYP-dependent EDHF" that has been demonstrated in large porcine coronary arteries [20] as well as in human mammary arteries [28], can be linked to the expression and activity of a CYP 2C epoxygenase [20,29] and inhibited by so-called EET antagonists [30]. From the results of the present investigation, it appears that the mechanisms that underlie EDHF-mediated responses vary within the vascular tree. Indeed, although CYP 2C was expressed in large porcine coronary arteries, a markedly weaker signal was obtained in small arteries (300–500 µm) isolated from the same hearts. Moreover, while the CYP inhibitors miconazole, clotrimazole, and sulfaphenazole attenuate EDHF-mediated responses in large coronary arteries [20], all three were without effect on the smaller vessels investigated in this study. EDHF-mediated relaxation of small coronary arteries was however sensitive to the combination of charybdotoxin and apamin as well as to the Na-K-ATPase inhibitor ouabain and therefore appears similar to the EDHF-mediated responses detected in human and porcine interlobar arteries [31,32].

Although a CYP epoxygenase was not implicated in EDHF-mediated relaxation of small porcine coronary arteries, experiments using a PLA2 inhibitor indicated that an endogenously generated prostanoid antagonized the EDHF-mediated relaxation. In addition, 17-ODYA and DDMS, a selective inhibitor of arachidonic acid {omega}-hydroxylation [14,33], mimicked the effect of PLA2 inhibition, suggesting the involvement of 20-HETE. Indeed, the exogenous application of 20-HETE markedly attenuated the bradykinin-induced, EDHF-mediated relaxation of U46619 [GenBank] -precontracted coronary arteries.

20-HETE can induce contraction by two mechanisms, one endothelium-dependent involving the cyclooxygenase-dependent generation of vasoconstrictor prostanoids, and the other endothelium-independent. The latter response is associated with inactivation of KCa+ channels [7,23], as well as the activation of Rho kinase, the phosphorylation of MLC20 and the sensitization of the contractile apparatus to Ca2+ [12]. Although we previously found no evidence to indicate that the 20-HETE-induced and Rho kinase-dependent contraction of small porcine coronary arteries involves the activation of this kinase [12], 20-HETE is reported to activate PKC in different cell types and thus influence KCa+ channel activity (for review, see Ref. [34]). The results of the present study clearly indicate that the inhibition of EDHF-mediated relaxation by 20-HETE also occurs via a PKC{alpha}-dependent process. Indeed, the effect of 20-HETE could be mimicked by PMA and prevented by a PKC inhibitor. Moreover, the activation of PKC by isometric stretch was not observed when the production of 20-HETE was prevented. Given that 20-HETE is reported to inhibit the Na-K-ATPase via a PKC-dependent mechanism and that the EDHF-mediated relaxation of small porcine coronary arteries is highly sensitive to ouabain, it is tempting to suggest that the functional antagonism of the EDHF-response occurs at the level of the ATPase.

Our report is not the first to suggest that coronary artery contraction antagonizes EDHF-mediated vasodilatation. Pressure-induced myogenic contraction of coronary arterioles, for example, has been reported to attenuate EDHF-mediated responses, most probably by interfering with KCa+ channel activation [35]. Whether this effect is related to the generation of 20-HETE was not determined but in canine basilar arteries, 20-HETE potentiates stretch-induced contraction via the PKC-mediated inhibition of KCa+ channels [23]. Both the latter observations and the results of the present investigation could reflect different steps in the same process.

It has been proposed that the production of 20-HETE can be attenuated by endothelium-derived NO. Indeed, the inhibition of endogenous 20-HETE formation by NO may account for some of the cyclic GMP-independent effects of NO on vascular tone [14,36]. However, although we previously reported that isometric stretch increases NO production [37], the fact that DDMS attenuated the U46619 [GenBank] -induced contraction of small porcine coronary arteries in the absence of a NO synthase inhibitor implies that, under the control conditions, the U46619 [GenBank] -induced contraction was associated with an increase in 20-HETE production although the NO synthase was active. Indeed, much of the evidence obtained to date that indicates that 20-HETE acts as a second messenger in the signaling cascades activated by endothelin-1 [38] and angiotensin II [39] were obtained in preparations/situations in which NO synthases were not inhibited.

Vascular disease states are generally associated with attenuated endothelium-dependent relaxation and a decrease in the bioavailability of NO. In such situations, EDHF has been proposed to act as a "back-up" vasodilator system. However, in situations associated with enhanced myogenic contraction or even coronary vasospasm, the increase in 20-HETE production, by antagonizing the EDHF-mediated relaxation, could severely compromise the vasodilator capacity of such vessels. It will therefore be interesting to determine whether coronary artery disease is associated with changes in the activity and/or expression of CYP 4A and whether CYP 4A represents a novel therapeutic target.


   Acknowledgements
 
Research described in this article was partly supported by Philip Morris, the Deutsche Forschungsgemeinschaft (FI 830/2-1), and the National Institute of Health (NIH GM31278, to JRF).


   Notes
 
Time for primary review 26 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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