Introduction

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Endothelin (ET)-1 is a 21-amino acid peptide and the most potent vasoconstrictor known. This peptide induces a sustained increase in renal vascular resistance that is associated with a marked decrease in renal blood flow (King et al., 1989). On a molar basis, its renal vasoconstrictor effect is greater than that produced by norepinephrine or angiotensin II (Ang II) (Rubanyi and Polokoff, 1994). ET can be produced locally, and in the rat kidney, specific high-affinity binding sites for ET have been localized to intrarenal arterial structures and glomeruli (Firth and Ratcliffe, 1992). Two functional ET receptors, ET_A and ET_B, have been identified by cloning and sequencing from a variety of species, including humans (Arai et al., 1990; Elshourbagy et al., 1993). Studies on the localization of ET receptor mRNA in the rat nephron using reverse transcription and the polymerase chain reaction assay indicated that the ET_B receptor mRNA distributes mainly in the collecting tubules and glomeruli, whereas small signals were found in the outer medullary collecting duct, cortical collecting duct, vasa recta, and arcuate artery. On the other hand, ET_A receptor mRNA was present primarily in the vascular system, namely, vasa recta and arcuate artery and in the inner medullary collecting duct, and the glomerulus (Terada et al., 1992). Evidence also suggests that mesangial cells possess both ET_A and ET_B receptor mRNA. Since the discovery of ET receptors, efforts have focused on identifying the functions mediated by each receptor. Where ET receptor-mediated functions were identified, the contribution of each subtype of receptor to the function is also being evaluated. Studies from various laboratories indicated that the ET_A receptor mediates the vasoconstrictor and growth effects of ET and that the ET_B receptor mediates the vasodilator and possibly some transport effects of ET (Brooks et al., 1994). Recent studies conducted in the rat, however, showed that the stimulation of ET_B receptors results in renal vasoconstriction (Clozel et al., 1992; Cristol et al., 1993; Pollock and Opgenorth, 1993).

The contribution of eicosanoids to ET-induced biological effects has been the subject of recent studies. ET stimulates phospholipases A₂ and C (Resink et al., 1989a,b; Simonson and Dunn, 1990), resulting in the release of free arachidonic acid (AA) from membrane phospholipid stores (Reynolds et al., 1989) and implicating oxygenase products of AA in some of the effects of ET. Cyclooxygenase (COX) products of AA metabolism were reported to mediate the bronchoconstrictor activity of ET in guinea pigs (Payne and Whittle, 1988), whereas lipoxygenase (LOX) products were demonstrated to contribute to the diuretic and natriuretic effects of ET-1 in the rat (Perico et al., 1991). The contribution of cytochrome P450 (CYP)-derived eicosanoids to the renal functional effects of ET-1 was unknown until recently, when we demonstrated that the renal vascular and tubular actions of ET-1 were greatly attenuated by inhibitors of the CYP pathway of AA metabolism (Oyekan et al., 1997; Oyekan and McGiff, 1998). Eicosanoids derived from CYP-AA metabolism are produced in renal blood vessels, including preglomerular microvessels (Imig et al., 1996), and may subserve a mediator/modulator role in the vasoactivity associated with various peptides and vasoactive hormones. In particular, afferent and interlobular arteries of the rat express mRNA and protein for a CYP4A2 enzyme and 20-hydroxyeicosatetraenoic acid (20-HETE), a major CYP-AA metabolite of the CYP4A2 family, potently constricted these vessels (Imig et al., 1996). In other studies, ET-1 and Ang II released 20-HETE and may account for their vasoconstrictor effects in the rat and rabbit kidney (Carroll et al., 1997; Oyekan et al., 1997).

In the present study, we evaluated the vasoconstrictor effects of ET-1 in preglomerular arterioles of the rat and characterized the receptors involved in relation to the contribution of HETE or epoxyeicosatetraenoic acid (EETs, epoxides) products of CYP metabolism of AA to this response. Our data suggest that both ET_A and ET_B receptors mediate the vasoconstrictor effect of ET-1 and that stimulation of both receptors appears to be coupled to 20-HETE release.

	Materials and Methods

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ET-1, ET-3, and BQ788 (N-cis-2,6-dimethyl-piperidino-carbonyl-L--methylleucyl-D-1-methoxycarbonyl-tryptophanyl-D-norleucine; Peninsula Laboratories, Belmont, CA) were stored in 0.1% acetic acid at 20°C. 12,12-Dibromododec-enoic acid (DBDD) and 20-HETE (gifts from Dr. Camille Falck, University of Texas South Western Medical Center), 17-octadecynoic acid (17-ODYA; BIOMOL Research Laboratories, Plymouth Meeting, PA), and 5,8,11,14-eicosatetraynoic acid (ETYA; Sigma Chemical Co., St. Louis, MO) were stored in ethanol at 70°C. BMS182874 [5-dimethylamino)-N-(3,4-dimethyl-5-isoxazolyl)-1-naphthalenesulfonamide] was dissolved in 0.1 M NaHCO₃, and miconazole (Sigma Chemical Co.) was dissolved in DMSO and stored at 4°C. Indomethacin (Sigma Chemical Co.) was freshly prepared in absolute ethanol.

Experiments were conducted on male Sprague-Dawley rats (Charles River, Wilmington, MA; body weight, 230 ± 8 g) according to protocols approved by the institutional animal care and use committee. The animals were placed in a room with lighting adjusted to produce a normal day/night cycle (illuminated from 8:00 AM to 8:00 PM). They were maintained on a standard rat food (Purina Chow) and were allowed ad libitum access to water and food before the experiments.

Isolated Microvessel Preparation. Male Sprague-Dawley rats were anesthetized with sodium pentobarbital (60 mg/kg i.p.). After a midventral laparotomy, the kidneys were flushed in situ via the abdominal aorta with 20 ml of cold (4°C) normal saline (0.9% NaCl). The kidneys were isolated and sectioned along the corticopapillary axis. Preglomerular arterioles, interlobular and arcuate [inner diameter (ID), 50-150 µm], were microdissected and mounted on glass micropipettes in a water-jacketed perfusion chamber in warmed (37°C), oxygenated (95% O₂/5% CO₂) Krebs-Henseleit buffer (PSS; containing 118 mM NaCl, 25 mM NaHCO₃, 1.9 mM CaCl₂, 1.19 mM MgSO₄, 4.75 mM KCl, 1.19 mM KH₂PO₄, and 11.1 mM dextrose, pH 7.2). The vessels were secured to the pipettes with 10-0 silk suture, and the side branches were tied off. The inflow pipette was connected to a pressurized reservoir to allow control of intraluminal perfusion pressure. The outflow cannula was clamped off, and intraluminal pressure was maintained at 80 mm Hg during the experiment. Vessels were allowed to equilibrate for 45 to 60 min, after which they were challenged with a submaximal dose of phenylephrine (PE; 10⁶ M). An arteriole was considered unacceptable for experimentation if it demonstrated leaks or failed to constrict to >20% to PE. Responses to ET-1 (4 × 10¹¹ to 2 × 10⁹ M), ET-3 (10¹¹ to 2 × 10⁹ M), or 20-HETE (10⁸ and 2 × 10⁸ M) were determined in Krebs' buffer containing vehicles for the inhibitors tested. Inhibitors were added to freshly prepared PSS, and a 30-min drug-tissue contact time was allowed before retesting the responses to the agonists in the same vessel. In all cases, PE was used as a negative control. Agonists were added to the bath (extraluminal application), and cumulative dose-response curves were generated, with 5 min between doses. After each dose-response relationship, the tissues were washed with fresh PSS for at least 30 min. Vascular diameters were measured 1 to 3 min after the addition of an agonist to the bath with the use of a video system composed of a stereomicroscope (Olympus BX40), a CCD television camera (model JE7826), a television monitor (Javelin), and a video measuring system (video micrometer, model JV6000T; Javelin). In time controls (n = 4), three successive cumulative dose-response relationships were established to ET-1, with a 30-min washout period allowed at the end of each dose-response curve.

Protocol. To determine whether ET_A and/or ET_B receptors are involved in the vasoconstrictor action of ET-1, ET-1-induced reduction in ID was evaluated in the presence of BMS182874 (n = 5, 3 µM), an ET_A receptor antagonist, or BQ788 (n = 5, 1 µM), an ET_B receptor antagonist. The response to ET-1 was also determined in vessels (n = 3) treated with both BMS182874 (3 µM) and BQ788 (1 µM).

Data Analysis. All responses were recorded as percentage of contraction (i.e., reduction in vessel diameter relative to baseline diameter before the addition of agonist). Data were expressed as mean ± S.E. ANOVA was used to compare dose-response curves between controls (vehicle-treated) and treated groups, followed by the Newman-Keuls test for dose-for-dose comparison. In all cases, P < .05 was considered significant.

	Results

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ET-1-Induced Vasoconstriction in Pressurized Preglomerular Arterioles. In interlobular and arcuate arteries pressurized to 80 mm Hg and superfused with Krebs' buffer at zero flow, ET-1 produced a dose-dependent reduction in ID, a measure of vasoconstriction (Fig. 1). The renal vasculature was more sensitive to ET-1 than PE as the threshold dose of ET-1 (4 × 10¹¹ M), which elicited a 12 ± 4% reduction in ID, is several orders lower than 10⁷ M, the threshold dose of PE. ET-1 at doses of 4 × 10¹¹, 2 × 10¹⁰, 4 × 10¹⁰, 10⁹, and 2 × 10⁹ M (n = 64) produced dose-dependent reductions in ID ranging from 25 ± 8 µm at 4 × 10¹¹ M to 142 ± 16 µm at 2 × 10⁹ M, corresponding to percent contractions ranging from 12 ± 4 to 86 ± 10%, respectively. Maximum contractions to ET-1 were obtained at a dose of 10⁹ M. Vasoconstrictor response to a submaximal dose of PE (10⁶ M) elicited a response (32 ± 4%) similar to that produced by 2 × 10¹⁰ M ET-1. In time controls (n = 4), responses to ET-1 were not different when the contractions were compared between the first and third dose-response relationships. For example, contractions to 10⁹ M ET-1 during the first dose-response relationship was 75 ± 11%, a value not different from that obtained (72 ± 5%) when the same dose of ET-1 was tested 120 to 130 min later.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Effects of ETA receptor antagonism with BMS182874 (a) or ETB receptor antagonism with BQ788 (b) or combined antagonism of ETA and ETB receptors with BMS182874 and BQ788 (c) on the vasoconstrictor response to ET-1. Effect of ET-1 (4 × 1011 to 2 × 109 M) on preglomerular arterioles (a and b, n = 5 vessels from 5 kidneys; and c, n = 3 vessels from 3 kidneys) under control conditions (CONTROL) and 30 min after BMS182874 (3 µM) and/or BQ788 (1 µM) was added to the bath. *, significant difference (P < .05) from control diameter measured at 80 mm Hg.

ET-1 Renal Vasoconstriction as Affected by ET_A or ET_B Receptor Antagonism. We demonstrated the involvement of ET_A and ET_B receptors in the renal vasoconstrictor response to ET-1, as BMS182874, the ET_A receptor antagonist, or BQ788, the ET_B receptor antagonist, blunted ET-1-induced vasoconstriction in the renal microvessels. BMS182874 (3 µM) attenuated ET-1-induced vasoconstriction in preglomerular vessels (n = 5) by 59 ± 4% (P < .05) (Fig. 1a). Thus, 4 × 10¹¹, 4 × 10¹⁰, and 2 × 10⁹ M ET-1 reduced ID by 5 ± 1%, 23 ± 10%, and 42 ± 13%, respectively, compared with reductions of 14 ± 2%, 53 ± 7%, and 84 ± 4% in vessels (n = 5) examined in vehicle-treated Krebs' buffer (P < .05) (Fig. 1a). In preglomerular vessels superfused with BQ788 (1 µM), the overall reduction in ET-1 effect was 50 ± 10% (P < .05). Thus, 4 × 10¹¹, 4 × 10¹⁰, and 2 × 10⁹ M ET-1 reduced ID by 8 ± 2%, 33 ± 4%, and 63 ± 8%, respectively, compared with reductions of 35 ± 11%, 70 ± 14%, and 86 ± 10%, respectively, in vessels (n = 5) superfused with vehicle (P < .05) (Fig. 1b). Further proof for the involvement of ET_A and ET_B receptors in the vasoconstrictor effect of ET-1 was obtained in vessels treated with both BMS182874 and BQ788, in which the reduction in ET-1 vasoconstriction was 75 ± 4% (P < .05), a value greater than that obtained with BMS182874 (59 ± 4%) or BQ788 (50 ± 10%) alone. ET-1 at 4 × 10¹¹, 4 × 10¹⁰, and 2 × 10⁹ M reduced ID by 8 ± 1%, 20 ± 4%, and 31 ± 10%, respectively, compared with 38 ± 7%, 82 ± 13%, and 91 ± 7%, respectively, in vessels (n = 3) treated with vehicle (Fig. 1c).

ET-1-Induced Renal Vasoconstriction as Affected by Inhibition of All Pathways of AA Metabolism. ETYA (10 µM), which inhibits the activities of all oxygenases metabolizing AA, COX, LOX, and CYP monooxygenase (MOX), attenuated the vasoconstrictor response to all doses of ET-1 (Fig. 2). ET-1 at doses of 4 × 10¹¹, 4 × 10¹⁰, and 2 × 10⁹ M reduced ID by 12 ± 4%, 38 ± 12%, and 67 ± 14%, respectively, when superfused with vehicle-treated Krebs' buffer, compared with contractions of 6 ± 4%, 28 ± 10%, and 45 ± 13%, respectively, in vessels (n = 5) treated with ETYA (10 µM) (Fig. 2). The same concentration of ETYA was without effect on PE-induced contraction (control, 45 ± 7%; experimental, 43 ± 13%).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effects of blockade of all pathways of AA metabolism with ETYA on the vasoconstrictor response to ET-1. Effect of ETYA on preglomerular vessels (n = 5 vessels from 5 kidneys) when vessels were exposed to vehicle (ethanol, final concentration 0.001%; CONTROL) or 30 min after addition of ETYA (10 µM) to the bath. *P < .05 versus CONTROL.

Effect of Inhibition of COX and CYP MOX on ET-1 Vasoconstriction. Indomethacin, a COX inhibitor, blunted ET-1-induced renal vasoconstriction. In preglomerular vessels (n = 7) treated with indomethacin (10 µM), 4 × 10¹¹, 4 × 10¹⁰, and 2 × 10⁹ M ET-1 reduced ID by 11 ± 3%, 43 ± 12%, and 67 ± 13%, respectively, compared with 22 ± 10%, 52 ± 11%, and 79 ± 9%, respectively, in vessels (n = 9) treated with vehicle (Fig. 3a). The inhibition by 10 µM indomethacin of ET-1 vasoconstriction was 28 ± 6% (P < .05), a value not different from that produced by the lower concentration (5 µM) of indomethacin (26 ± 6%, P < .05; n = 9) (data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Effects of inhibition of COX with indomethacin (INDO; a), 20-HETE production with DBDD (b) or 17-ODYA (c), or combined inhibition of COX and 20-HETE production with indomethacin and DBDD (INDO/DBDD; d) on the vasoconstrictor response to ET-1. Effect of ET-1 (4 × 1011-2 × 109 M) on preglomerular arterioles (a, n = 9 vessels from 7 kidneys; b, n = 5 vessels from 5 kidneys; c, n = 6 vessels from 5 kidneys; and d, n = 3 vessels from 3 kidneys under control conditions (CONTROL) and 30 min after indomethacin (10 µM; a), DBDD (2 µM; b), 17-ODYA (2 µM; c) or indomethacin (10 µM) and DBDD (2 µM) (d) were added to the bath. *P < .05 versus CONTROL.

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20-HETE Vasoconstriction and Role of COX. 20-HETE (10⁸ and 2 × 10⁸ M) elicited a potent dose-related vasoconstriction, reducing ID by 16 ± 3 and 31 ± 5 µm, respectively, which corresponded to contractions of 12 ± 2 and 23 ± 4%, respectively. These responses were 3 orders weaker compared with corresponding doses of ET-1. Superfusion with indomethacin (5 µM; n = 8) was without effect on 20-HETE vasoconstriction. Reductions in ID by 10⁸ and 2 × 10⁸ M ET-1 were 13 ± 5 and 23 ± 6%, respectively, in vessels (n = 5) treated with indomethacin (5 µM), values not different from those obtained in vessels contracted with 20-HETE in vehicle-treated buffer (n = 8) (Fig. 4). However, at the higher concentration of indomethacin (10 µM; n = 5), 20-HETE vasoconstriction was attenuated by 55 ± 3% (P < .05); the reductions in ID by 10⁸ and 2 × 10⁸ M 20-HETE were 4 ± 2 and 13 ± 4%, respectively, compared with reductions of 12 ± 2 and 23 ± 4%, respectively, in vessels (n = 6) contracted with 20-HETE in vehicle-containing buffer (Fig. 4). The same concentration of indomethacin was without effect on the vasoconstrictor responses to 10⁶ M PE (control, 34 ± 3%; experimental, 31 ± 4%).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Effect of indomethacin (INDO) on the dose-related vasoconstrictor effect of 20-HETE (108 and 2 × 108 M) on preglomerular vessels. Shown in the figure is the effect of indomethacin (5 and 10 µM) under control conditions (CONTROL) and 30 min after addition of indomethacin to the bath. n refers to number of vessels/kidneys. *P < .05 versus CONTROL.

ET-1 Vasoconstriction as Affected by Inhibition of 20-HETE and ET_A or ET_B Receptor Antagonism. We obtained evidence that the CYP-dependent vasoconstrictor response to ET-1 is coupled to both ET_A and ET_B receptors in the preglomerular renal vessel of the rat. In vessels superfused with the combination of BMS182874 (3 µM) and 17-ODYA (2 µM), ET-1 reduced ID by 64 ± 2% (P < .05), a value not different from that obtained with BMS182874 (59 ± 4%; Fig. 1a) alone. In vessels treated with BMS182874 and 17-ODYA (n = 5), 4 × 10¹¹, 4 × 10¹⁰, and 2 × 10⁹ M ET-1 reduced ID by 5 ± 2, 13 ± 2, and 16 ± 2%, respectively, compared with 12 ± 3, 30 ± 3, and 45 ± 8%, respectively, in vehicle-treated (n = 5) vessels (Fig. 5a). Similarly, the combination of BQ788 (1 µM) and 17-ODYA (2 µM) (n = 5) blunted the reduction in ID to the same doses of ET-1 by 4 ± 1, 22 ± 11, and 32 ± 14%, respectively, compared with 8 ± 2, 40 ± 15, and 57 ± 17%, respectively, in vessels (n = 5) superfused with vehicle (Fig. 5b). The effect of BQ788 and 17-ODYA amounted to 45 ± 6% inhibition of ET-1 vasoconstriction, a value not different from that obtained with 17-ODYA alone (50 ± 6%; Fig. 3c). Further support for an ET_B-coupled CYP-dependent ET-1 vasoconstriction was obtained in vessels (n = 3) contracted with ET-3, an ET_B-selective agonist, in the presence of 17-ODYA. Under control conditions, 2 × 10¹⁰, 10⁹, and 2 × 10⁹ M ET-3 reduced ID by 7 ± 2, 16 ± 3, and 23 ± 7%, respectively, whereas 17-ODYA attenuated these responses to 5 ± 2, 11 ± 3, and 13 ± 3%, respectively (P < .05).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effects of combined blockade of 20-HETE production with 17-ODYA and ETA receptor antagonism with BMS182874 (a; 17-ODYA/BMS182874) or ETB receptor antagonism with BQ788 (b; 17-ODYA/BQ788) on vasoconstrictor response ot ET-1. Effect of 17-ODYA on vasoconstrictor response to ET-3 (17-ODYA) is presented in c. Preglomerular vessels (a and b, n = 5 vessels from 5 kidneys; c, n = 3 vessels from 3 kidneys) were superfused for 30 min with 17-ODYA (2 µM), BMS182874 (3 µM), or BQ788 (1 µM) before testing the vasoconstrictor effect of ET-1 (a and b) or ET-3 (c). *P < .05 versus CONTROL.

	Discussion

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The findings reported in the present study extend our previous observations (Oyekan et al., 1997; Oyekan and McGiff, 1998) and confirm that CYP-dependent /-1 hydroxylase-derived eicosanoids make a major contribution to the renal functional response to ET-1 in the rat. Thus, ET-1 produced potent reduction in ID of the pressurized preglomerular vessel. This effect was reduced by inhibition of all of the pathways of AA metabolism, inhibition of COX, and inhibition of 20-HETE production but was not affected by inhibition of epoxides. Moreover, ET-1-induced vasoconstriction was inhibited by ET_A or ET_B receptor antagonism and inhibition of 20-HETE production. These data suggest that both ET_A and ET_B receptors mediate the vasoconstrictor effect of ET-1 in the renal microvessel and that both receptors are coupled to 20-HETE, a major CYP-AA metabolite that constricts isolated blood vessels and perfused vascular beds.

We recently linked ET-1 to renal production of CYP-AA metabolites in the rat isolated kidney; namely, ET-1 released 20-HETE associated with renal vasoconstriction. Furthermore, inhibition of CYP MOX activity reduced the vasoconstrictor activity of ET-1 by 40% while decreasing renal efflux of 20-HETE (Oyekan et al., 1997). However, the ET receptors involved in this effect have not been demonstrated. Generally, the question of whether renal vasoconstriction by ET is mediated primarily by an ET_A or ET_B receptor has not been clarified. The issue is compounded by species differences. In the dog, for example, the renal vasoconstrictor effect of ET-1 was reported to be mediated through ET_A receptors as ET-1 vasoconstriction was inhibited by BQ123, an ET_A-selective antagonist, and S6c, an ET_B-selective agonist, was without effect (Brooks et al., 1994), However, in the rat, S6c vasoconstricted the renal vasculature, whereas BQ123, an ET_A-selective antagonist, did not block ET-1 vasoconstriction (Cristol et al., 1993; Pollock and Opgenorth, 1993). This led to the conclusion that ET-1 may cause renal vasoconstriction in the rat by stimulating ET_B receptors. This conclusion was soon challenged by the studies of Pollock and Opgenorth (1994), in which they observed that BQ123 blocked the renal vasoconstriction of lower doses of ET-1 and its precursor, big-ET, and suggested that a third type of receptor, with a lower affinity to ET-1, may be involved in the vasoconstrictor effect of high doses of ET-1 in the rat kidney.

Recent experimental evidence suggests that in addition to the classic ET_A receptor-related vasoconstriction, renal vascular effects of ET-1 may be mediated by a subgroup of ET_B receptors, the ET_B2 subtype, mediating vasoconstriction, as opposed to the vasodilator ET_B1 receptors (Warner et al., 1993). However, ET-1 has also been reported to produce ET_A-related renal cortical vasoconstriction but an ET_B-mediated renal medullary vasodilation in the rat (Gurbanov et al., 1996; H.C.H. and A.O.O., unpublished observation). The present study unequivocally shows that both ET_A and ET_B receptors mediate renal vasoconstriction in the rat microvessel inasmuch as BMS182874, an ET_A receptor antagonist, or BQ788, an ET_B receptor antagonist, inhibited ET-1 vasoconstriction. Further evidence for mediation by both receptors was provided by the observation that ET-3, an ET_B-selective agonist, elicited a vasoconstrictor effect. Moreover, the additive inhibition observed when ET-1 vasoconstriction was evaluated in the presence of both antagonists supports the involvement of ET_A and ET_B receptors as inhibition of ET-1 effect increased to ~70% from individual values of ~50% with each antagonist.

Postreceptor cellular signaling mechanisms by peptides of the ET gene family demonstrated that ET stimulates phospholipases A₂ and C in vascular smooth muscle cells (Resink et al., 1989a,b; Simonson and Dunn, 1990), causing the release of AA (Reynolds et al., 1989), which can undergo transformation via any of the three oxygenases. A role has therefore been proposed for COX- and LOX-derived eicosanoids in the effects of ETs in the kidney (Perico et al., 1991; Gurbanov et al., 1996) and in the vascular smooth muscle. In the porcine pial arteriole, the rat liver microcirculation, and the lungs, vasoconstriction to ET was shown to be partially prostanoid-dependent, inasmuch as it was suppressed by inhibitors of COX or thromboxane A₂ synthase (Payne and Whittle, 1988; Armstead et al., 1989; Kurihara et al., 1992). Our data support a role for COX-derived eicosanoids in ET-1 vasoconstriction in the renal arteriole of the rat as indomethacin inhibited the response (Fig. 3). These findings are in agreement with other studies (Eglen et al., 1989; Asano et al., 1994) and our previous study (Oyekan et al., 1997).

A role for CYP-dependent /-1 hydroxylase-derived eicosanoids in ET-1-induced renal vasoconstriction in the rat renal microvessel is supported by the attenuation of this effect by 17-ODYA or DBDD, inhibitors of 20-HETE production. We have previously linked ET-1-induced renal effects to the production of CYP-AA metabolites, especially 20-HETE in the isolated perfused kidney (Oyekan et al., 1997), in the anesthetized rat (Oyekan and McGiff, 1998), and in the unanesthetized hypertensive rat treated with deoxycorticosterone acetate-salt (Oyekan et al., 1999). In the latter study, increased 20-HETE excretion occurred in parallel with ET-1 excretion, suggesting that endogenous production of 20-HETE is related to ET-1 production, favoring a proposed role for 20-HETE as a second messenger in the renal effects of ET-1. 20-HETE, a product of the CYP4A2 family, is produced in the renal microvessel (Imig et al., 1994) and can be stored in tissue lipids (Karara et al., 1991), where it can be released in response to hormonal stimuli as, for example, Ang II-stimulated, receptor-mediated hydrolysis of lipids (Carroll et al., 1997). Data from the present study suggest that 20-HETE contributes to vasoconstriction of ET-1 and ET-3 in the rat renal microvessel, as demonstrated by inhibition of the vasoconstriction by 17-ODYA and/or DBDD, inhibitors of 20-HETE production.

In the isolated blood vessel, 20-HETE elicits an endothelium- and COX-dependent vasoconstriction that is ascribed to its endoperoxide product, 20-hydroxy-prostaglandin H₂ (Escalante et al., 1989; Schwartzman et al., 1989). However, in the rat renal arteriole, 20-HETE was reported to evoke a COX-independent vasoconstriction (Imig et al., 1994). Our data are at variance with the latter study in that in our hands, the same concentration of indomethacin (10 µM) used by Imig et al. (1996) inhibited 20-HETE vasoconstriction by ~30%. Although indomethacin can interfere with Ca²⁺ fluxes across cell membranes and thereby cause nonspecific effects, this does not seem to be the case as the vasoconstriction elicited by PE was not affected at the same concentration of indomethacin. Our data, therefore, support that 20-HETE vasoconstriction in the preglomerular arteriole is COX-dependent, an observation similar to that in isolated blood vessels (Escalante et al., 1989; Schwartzman et al., 1989) and perfused kidney (Askari et al., 1997). This being so, the differential in the degree of inhibition by indomethacin of ET-1 effect (~26%) compared with that obtained with 17-ODYA (~60%) or DBDD (~50%) was unexpected. This observation suggests that the contribution of the CYP MOX pathway via 20-HETE to ET-1 vasoconstriction is not exclusively linked to the COX pathway and casts some doubt on whether all of the vasoconstriction produced by 20-HETE is mediated via COX-transformed products. The observation that 20-HETE vasoconstriction was unaffected by indomethacin at a low concentration (5 µM) and was not totally inhibited at the higher concentration (10 µM) suggests that 20-HETE vasoconstriction is not totally mediated via COX products. If, indeed, 20-HETE, the putative mediator of ET-1 renal vasoconstriction, was further metabolized by COX to vasoconstrictor intermediates, there should be no difference in the inhibition of ET-1 effect in the presence of indomethacin and DBDD. However, we observed an inhibition of ET-1 vasoconstriction in the presence of the combination of indomethacin and DBDD that was greater than that produced by either agent alone. This suggests that the effects of these inhibitors are independent and distinct, and therefore, the involvement of COX and CYP MOX pathways in ET-1 renal vasoconstriction are not mutually exclusive. However, considering that the overall effect by this combination was greater than that produced by ETYA, which inhibits all pathways of AA metabolism, the results are suspect and are difficult to interpret in regard to the role of eicosanoids in this effect. Moreover, the fact that PE-induced vasoconstriction was not affected under the same experimental condition suggests that this is a nonspecific effect.

In conclusion, we have presented evidence to demonstrate that both ET_A and ET_B receptors mediate renal vasoconstriction to ET-1 in the rat renal arteriole. The activation of both receptors is linked to 20-HETE production inasmuch as the effect of combined inhibition of 20-HETE production and ET_A or ET_B receptor antagonism was not different from that obtained with either antagonist or inhibitor alone. Moreover, COX-derived eicosanoids contribute to ET-1 vasoconstriction, and 20-HETE-induced vasoconstriction was also COX-dependent. Further work continues to clarify whether CYP MOX and COX pathways are independent pathways in ET-1 vasoconstriction.