Angiotensin II releases 20-HETE from rat renal microvessels

Kevin D. Croft1, John C. McGiff2, Alicia Sanchez-Mendoza3, and Mairead A. Carroll2

2 Department of Pharmacology, New York Medical College, Valhalla, New York 10595; 1 Department of Medicine, University of Western Australia, Perth WA 6847, Australia; and 3 Centro de Investigacion y Estudios Avanzados Del Instituto Politécnico Nacional, Mexico City, Mexico 07000


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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We studied hydroxyeicosatetraenoic acid (HETE) release in response to ANG II from preglomerular microvessels (PGMVs), the vascular segment governing changes in renal vascular resistance. PGMVs were isolated from Sprague-Dawley rats and incubated with NADPH and hormones at 37°C. Eicosanoids were extracted, and cytochrome P-450 (CYP)-derived HETEs were purified and quantitated by negative chemical ionization gas chromatography-mass spectroscopy. PGMVs produced primarily 20- and 19-HETEs, namely, 7.9 ± 1.7 and 2.2 ± 0.5 ng/mg protein, respectively. ANG II (5 nM) increased CYP-HETE release by two- to threefold; bradykinin, phenylephrine, and Ca2+ ionophore were without effect. [Sar1]ANG II (0.1-100 µM) dose dependently stimulated 19- and 20-HETEs, an effect blocked by the AT2-receptor antagonist PD-123319 as well as by U-73122, a phospholipase C inhibitor. Microvascular 20-HETE release was increased more than twofold by the third day in response to ANG II (120 ng · kg-1 · min-1) infused subcutaneously for 2 wk; it was not further enhanced after 14 days, although blood pressure continued to rise. Thus an AT2-phospholipse C effector unit is associated with synthesis of a vasoconstrictor product, 20-HETE, in a key renovascular segment.

kidney; arachidonic acid metabolism; angiotensin II receptors; arteries; gas chromatography-mass spectroscopy


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INTRODUCTION
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ANG II STIMULATES eicosanoid synthesis by receptor-mediated activation of lipases to release arachidonic acid (AA) that is metabolized by cyclooxygenases (COX), lipoxygenases, and cytochrome P-450 monooxygenases (CYP) (38) in a cell/tissue specific manner. These arachidonate products demonstrate a wide range of biological activities of special relevance to renal function. Arachidonate products of 12- and 15-lipoxygenases are second messengers for ANG II (45), as has been demonstrated for the renal hemodynamic effects of the peptide (40). Under physiological conditions, PGE2 and PGI2 modulate the renal vascular and tubular responses to ANG II, whereas under pathophysiolgical conditions PGH2 and thromboxane A2 intensify the vasoconstrictor and antinatriuretic effects of ANG II, as reported in angiotensin-dependent forms of hypertension (38). ANG II increases renal synthesis of CYP-derived AA products, hydroxyeicosatetraenoic acids (HETEs), and epoxyeicosatrienoic acids (EETs; epoxides), which modulate and mediate the actions of the peptide in the renal vasculature and the nephron (7). Preformed HETEs and EETs are also released by ANG II from storage sites, mainly phospholipids, by peptide activation of acylhydrolases, chiefly phospholipase A2 (PLA2) (6). ANG II produced a several-fold increase in the efflux of 20-HETE and subterminal HETEs (16-, 17-, 18-, and 19-HETEs) from the rabbit kidney, whereas an equipressor concentration of arginine vasopressin did not increase efflux of HETEs (7). The biological activities of 16-, 17-, 18- and 19-HETEs are potentially important in view of their effects on blood vessels and Na+-K+-ATPase activity (7). 20-HETE occupies a preeminent position in renal regulatory mechanisms: for the tubules, by modulating the activities of the tubular Na+-K+-2Cl- cotransporter (14), Na+-K+-ATPase (9) and K+ channels (51); and for the vasculature, by mediating tubuloglomerular feedback (56) and autoregulation of blood flow (55).

The AT2 receptor, which exhibits only 30% homology with the AT1 receptor, is widely distributed in fetal tissues. In most tissues, its expression diminishes rapidly after birth (17), so that in the adult rat it accounts for ~5% of the renal AT receptors. However, the AT2 receptor may be expressed in adult tissues during cell proliferation and differentiation (46, 47), as in the response to vascular injury (27), and is upregulated by sodium depletion and ANG II (39, 43). The predominant ANG II receptor subtype in adult rat and human kidneys is the AT1, accounting for 90-95% of total renal ANG II receptors; it is prominent in glomeruli, proximal tubules, the inner stripe of the outer medulla, and vasa recta (53). Although AT1 receptors predominate in most adult tissues and most of the known effects of ANG II on renal function can be attributed to the AT1 receptor, the preglomerular arteries and arterioles may be an exception (16, 19, 50). In rat interlobular arteries, AT2 receptors may participate in the regulation of vascular tone, as AT2 receptor blockade partially reversed the renal vasoconstrictor response to ANG II (19). AT2 receptors are also present in afferent arterioles (1). After blockade of AT1 receptors, ANG II produced dose-dependent dilatation of the rabbit afferent arteriole that could be prevented by either blockade of AT2 receptors or inhibition of synthesis of EETs.

To localize critical sites of ANG II-CYP interactions intrarenally, and to define the mechanism by which ANG II stimulates CYP-derived HETE synthesis, we studied HETE release in response to ANG II from preglomerular microvessels (PGMVs), the vascular segments governing changes in renal vascular resistance (49). PGMVs generate relatively large quantities of 20-HETE and lesser amounts of EETs. We designed the present experiments to define 1) the CYP-derived AA product involved in the response to ANG II stimulation of the preglomerular vasculature; 2) the ANG II receptor subtype involved in release of HETEs from PGMVs; and 3) the acylhydrolase(s) stimulated by ANG II to release AA for conversion to CYP-related AA products. We found, unexpectedly, that AT2 receptors mediated ANG II-induced release of CYP-derived AA products, primarily 20-HETE, from rat PGMVs. Furthermore, release of 20-HETE in response to stimulation of AT2 receptors was linked to activation of phospholipase C (PLC).


    MATERIALS AND METHODS
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Isolation and identification of rat kidney PGMVs. Male Sprague-Dawley rats (300-320 g; Charles River) were anesthetized with sodium pentobarbital (100 mg/kg, ip). After laparotomy, the kidneys were isolated and flushed, via the abdominal aorta, with 20 ml saline to remove blood elements, followed immediately with 20 ml of a 5% solution of iron oxide. The kidneys were removed and cut in half along the corticopapillary axis. The cortex was excised and finely diced with a razor blade at 4°C. The minced cortex was homogenized in ~20 ml phosphate-buffered saline (Sigma). The iron laden PGMVs were separated from other cortical tissues, e.g., tubules, by magnetic separation (Advanced Magnetics). After several magnetic isolations, the PGMVs were passed through an 18-g needle and subsequently through 21- and 23-g needles to remove attached tubules and glomeruli, and the resultant suspension was subjected to a final magnetic separation. The PGMVs were washed three times in Tyrode's solution (Sigma) containing indomethacin (10 µM), gassed with 95% O2-5% CO2. The composition of the Tyrode solution was (in g/l) 8 NaCl, 0.2 KCl, 0.1002 MgCl2, 0.2 CaCl2, 0.05 NaHPO4, 1.0 NaHCO3, and 1.0 glucose, pH 7.4. Indomethacin (10 µM) was included to inhibit metabolism of CYP-AA metabolites by COX.

The purity of each microvascular preparation was examined by using light microscopy (×400 magnification). The vessel diameters were measured in several preparations by using a linear eyepiece reticle (0.05-mm divisions, Fischer Scientific, Springfield, NJ). Only preparations that had minimal proximal tubular contamination (<5%) were used for the experiments. Furthermore, a marker for proximal tubules, alkaline phosphatase activity, was measured as described previously (33). Briefly, an aliquot of the microvessel suspension was incubated with 14 mM p-nitrophenylphosphate in a substrate buffer (pH 10.5) containing 0.1 M glycine and 1 mM MgCl2 for 30 min at 37°C. The reaction was stopped with 50 mM NaOH, and the absorbance of the product formed, p-nitrophenol, was measured at 405 nm. As a positive control, alkaline phosphatase activity was measured in aliquots of cortical homogenates from which the PGMVs had been isolated. These samples contained high numbers of proximal tubules. Protein concentration was determined by using the Bradford method (4).

Quantitation of CYP-HETEs. To quantitate basal and stimulated CYP-HETEs released by PGMVs, suspensions of PGMVs (~0.5 mg protein/ml) were incubated in the presence and absence of NADPH (1 mM) and agonists at 37°C for 1-30 min. Enzyme inhibitors and receptor antagonists were added to incubates 5-10 min before addition of ANG II. The PGMVs and media eicosanoids were extracted after addition of 2 ng deuterated (D2) 20-HETE as an internal standard. The CYP-HETEs were purified by using reverse phase (RP)-HPLC and quantitated by negative chemical ionization gas chromatography-mass spectroscopy (GC-MS).

Quantitation of esterified EETs and HETEs. The PGMVs were homogenized in methanol-water (1:1), and lipids were extracted by using the Bligh/Dyer method (3). The fractions were subjected to alkaline hydrolysis by incubating with 200 µl methanol and 100 µl potassium hydroxide (0.2 M) for 2 h at 50°C (6). Eicosanoids were extracted after addition of 2 ng D2 20-HETE and 2 ng D8 11,12-EET as internal standards. The CYP-HETEs and EETs were purified by using RP-HPLC and quantitated by negative chemical ionization GC-MS.

Purification of CYP-HETEs. The samples were extracted twice with 2 vol acidified ethyl acetate (pH 4.0) and evaporated to dryness. The samples were purified by RP-HPLC on a C18 µBondapak column (4.6 × 24 mm) by using a linear gradient from acetonitrile- water-acetic acid (62.5:37.5:0.05%) to acetonitrile (100%) over 20 min at a flow rate of 1 ml/min. Fractions containing CYP-HETEs were collected on the basis of the elution profile of standards monitored by ultraviolet absorbance (205 nm). The fractions were evaporated to dryness and derivatized for GC-MS analysis. Radiolabeled samples were extracted and separated as described above, and radioactivity was monitored by an on-line radio detector (beta -RAM, INUS Systems).

Derivatization and mass spectrometric analyses. Pentafluorobenzyl esters were prepared by the addition of alpha -bromo-2,3,4,5,6-pentafluorotoluene (pentafluorobenzylbromide; 5 µl, Aldrich) and N,N-diisopropylethylamine (5 µl, Aldrich) to a sample dissolved in acetonitrile (100 µl), and the derivatization was continued at room temperature for 30 min (32). Trimethylsilyl ethers of hydroxyls were prepared by dissolving the sample in N,O-bis(trimethylsilyl)trifluoroacetamide (80 µl; Sigma), and the reaction was continued at room temperature for 30 min. To separate subterminal CYP-HETEs, samples were dissolved in isooctane, and 1-µl aliquots were injected into a GC (HP-5890) column (DB-1; 15.0 m; 0.25 mm, inner diameter: 0.25 µm film thickness, Supelco) by using a temperature program ranging from 150 to 300°C at a rate of 10°C/min (32). Methane was used as a reagent gas at a flow resulting in a source pressure of 1.3 Torr, and the MS (Hewlett-Packard 5989A) was operated in electron capture chemical ionization mode. The endogenous CYP-HETEs were identified [ion mass-to-charge ratio (m/z) 391] by comparison of GC retention times with authentic CYP-HETE standards synthesized by us and quantitated by calculating the ratio of abundance with D2 20-HETE (m/z 393). The endogenous EETs were identified (ion m/z 319) by comparison of GC retention times with authentic D8 11,12-EET (m/z 327) standard (Biomol).

In vivo treatment with ANG II. To study the temporal regulation of ANG II treatment on microvascular CYP-HETE release, we infused ANG II for either 3 or 14 days and monitored changes in blood pressure and determined differences in CYP-HETE release between PGMVs obtained from treated and control rats. Sprague-Dawley rats (300-320 g) were anesthetized with methoxyflurane (50 mg/kg) and miniosmotic pumps (Alzet; 0.5 µl/h) were implanted subcutaneously in the nape of the neck. The osmotic pumps contained either ANG II (120 ng · kg-1 · min-1) or 0.01 N acetic acid (vehicle control). Systolic blood pressure (tail cuff) was monitored before implantation and on the day of microvessel purification. PGMVs were isolated as described above.

Statistical analysis. Comparisons among several groups were made by analysis of variance followed by a modified t-test. Paired analyses were used when comparisons were made of data obtained from the same experimental preparation (i.e., basal and stimulated levels). Data are expressed as means ± SE, and a P value of <0.05 was considered significant.


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Microscopic and biochemical identification of renal PGMVs. The purity of each microvascular preparation was examined by using light microscopy (×400 magnification). Measurement of PGMV diameters, using an eyepiece linear reticle, revealed the presence of interlobar (135 ± 9 µm) and arcuate (74 ± 5 µm) arteries, and interlobular (42 ± 1 µm) and afferent (14 ± 1 µm) arterioles. On the basis of the number and size of each vessel, we assessed the relative contribution of larger PGMVs (interlobar and arcuate arteries) to smaller PGMVs (interlobular and afferent arterioles) as ~50%:50%. Furthermore, measurement of alkaline phosphatase activity as an index of proximal tubule contamination disclosed undetectable activity in microvessel incubates in which HETEs were measured. However, in concentrated samples, i.e., 10 times more concentrated than our experimental incubates, 5.0 ± 1.3 nmol p-nitrophenol · mg protein-1 · min-1 was detected. Alkaline phosphatase activity measured in aliquots of cortical homogenates, i.e., the residue from which the PGMVs had been isolated, formed 814.4 ± 130.9 nmol p-nitrophenol · mg protein-1 · min-1. The latter samples contained an abundance of proximal tubules on the basis of microscopic evaluation.

Quantitation of CYP-HETEs by renal PGMVs. Renal PGMVs produced primarily 20-HETEs with lesser amounts of 19-HETE and negligible amounts of 16-; 17- and 18-HETEs (Fig. 1). Basal formation of 20- and 19-HETEs was time-and NADPH-dependent. The ratio of 20- to 19-HETE varied from 6 to 2:1 over a time course of 1-20 min (Fig. 2). We performed a total lipid extraction on a concentrated sample of PGMVs (~10 mg protein) to quantitate any esterified HETEs. However, unlike cortical tissue (6) there was no evidence of esterified HETEs in the purified microvasculature. As there was a possibility that the isolation and purification of the PGMVs activated phospholipases and released esterified HETEs, we also assayed for EETs in the same PGMV samples (n = 3). By adding 2 ng of D8 11,12-EET, we determined that the PGMVs contained 1.0 ± 0.2 ng of esterified EETs/mg protein. The absence of esterified HETEs, therefore, was not likely related to their prior release.


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Fig. 1.   A representative negative chemical ionization gas chromatography-mass spectroscopy (GC-MS) analysis of cytochrome P-450-hydroxyeicosatetraenoic acids (CYP-HETEs) released by rat renal preglomerular microvessels (PGMVs). Derivatized CYP-HETEs were analyzed by single ion monitoring at m/z 391 (endogenous metabolites and authentic standards) or at m/z 393 (internal standard D2 20-HETE). Top: retention times of endogenous microvessel metabolites. Middle: D2 20-HETE internal standard (4.34 min.). Bottom: retention times of authentic standards, 16-; 17-; 18-; 19- and 20-HETE.



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Fig. 2.   Time-dependent release of 20- and 19-HETEs from rat renal PGMVs. PGMVs were incubated with NADPH (1 mM) for 1-20 min. After lipid extraction, the CYP-HETEs were purified, derivatized and quantitated by using GC-MS. Values are means ± SE; n = 4. * P < 0.05.

During a 5-min incubation, ANG II (5 nM) increased 20-HETE release from PGMVs by twofold and was without effect on 19-HETE release (Fig. 3). Stimulation of HETE release by ANG II was specific as several vasoactive agonists including phenylephrine (1 µM), bradykinin (1 µM), and endothelin-1 (1 µM) did not release HETEs (Fig. 4). Furthermore, HETE release was unaffected by angiotensin 1-7 (10-6 M), which has been shown to stimulate renal prostaglandin production (11), nor by the Ca2+ ionophore (10 µM). Although ANG II (5 nM-1 µM) dose dependently stimulated HETE release, the threshold dose varied greatly, from 5 to 100 nM, presumably related to the level of activity of tissue peptidases that degraded ANG II. The results shown in Fig. 5 were obtained by using [Sar1]ANG II, a stable analog of ANG II, which has been used in proximal tubule studies (26). Release of 19- and 20-HETEs was stimulated in a dose-dependent manner by [Sar1]ANG II.


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Fig. 3.   Stimulated release of renal microvascular CYP-HETEs (20- and 19-HETEs) after incubation with 5 nM ANG II for 5 min. Incubates were extracted and the CYP-HETEs were purified, derivatized, and quantitated by using GC-MS. Values are means ± SE; n = 4. * P < 0.05 compared with control value.



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Fig. 4.   Release of renal microvascular 20-HETE after incubation with various vasoactive hormones for 5 min. Incubates were extracted and the CYP-HETEs were purified, derivatized, and quantitated by using GC-MS. ANG II (1 µM); bradykinin (BK; 1 µM); phenylephrine (PE; 1 µM); endothelin-1 (ET-1; 1 µM). Values are means ± SE; n = 3. * P < 0.05 compared with control value.



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Fig. 5.   Dose-dependent release of 20- and 19-HETE in response to [Sar1]ANG II. Rat renal PGMVs were incubated with various concentrations of [Sar1]ANG II and NADPH (1 mM) for 5 min. After lipid extraction, the CYP-HETEs were purified, derivatized, and quantitated by using GC-MS. Values are means ± SE; n = 6. * P < 0.05 compared with control value.

ANG II receptor subtype linked to stimulation of HETE release. PGMVs were incubated with [Sar1]ANG II (10 µM) in the presence and absence of either losartan (DUP-753; 100 µM), an AT1-receptor antagonist, or PD-123319 (100 µM), an AT2-receptor antagonist. The increase in 20-HETE release produced by [Sar1]ANG II (10 µM) was unaffected by losartan but was inhibited by PD-123319 (Fig. 6). Furthermore, a 10-fold lower concentration of PD-123319 (10 µM) reduced stimulated 20-HETE release by 30% (data not shown) compared with the 55% reduction in 20-HETE with the higher PD-123319 (100 µM) concentration shown in Fig. 6. The receptor antagonists did not affect basal HETEs release. Although changes in 19-HETE release paralleled those of 20-HETE in the presence of the ANG II-receptor antagonists, they did not attain significance (Fig. 6).


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Fig. 6.   Effect of ANG II-receptor antagonists on renal microvascular CYP-HETE release. PGMVs were incubated for 5 min with and without [Sar1]ANG II in the presence of an AT1-receptor antagonist, DUP-753, and an AT2 receptor antagonist, PD-123319. Incubates were extracted and the CYP-HETEs were purified, derivatized, and quantitated by using GC-MS. A: 20-HETE data. B: 19-HETE data. Values are means ± SE; n = 4. * P < 0.05 compared with control value.

Characterization of phospholipase coupled to CYP-HETE release. To address the regulation of ANG II-stimulated HETE formation and release, the phospholipase responsible for providing AA substrate to CYP omega /omega -1 hydroxylases was examined. PGMVs were incubated with [Sar1]ANG II (10 and 100 µM) in the presence and absence of inhibitors of PLA2 and PLC. The PLC inhibitor, U-73122 (10 µM), prevented 20-HETE release in response to [Sar1]ANG II (Fig. 7), whereas U-73343, the inactive structural analog of U-73122, was without effect. None of the PLA2 inhibitors that we studied affected either [Sar1]ANG II-stimulated or basal levels of microvessel CYP-HETE release (Table 1). Neither inhibitors of cytosolic or secretory PLA2 [arachidonyl trifluoromethyl ketone, 1 µM; aristolochic acid, 100 µM; 2-(pp-amylcinnamoyl)amino-4-chlorobenzoic acid, 0.5 µM; palmityl trifluromethyl ketone, 20 µM; haloenol lactone suicide substrate, 100 nM] affected microvascular [Sar1]ANG II-stimulated release of 20-HETE. In addition, oleyloxyethyl phosphorylcholine (1 µM), a potent and specific substrate analog PLA2 inhibitor that abolished the myogenic response in dog arcuate arteries mediated by 20-HETE (28), also was without effect on microvascular HETE release.


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Fig. 7.   Phospholipase C inhibitor (U-73122; 10 µM) prevented [Sar1]ANG II-stimulated release of 20-HETE from rat renal PGMVs. PGMVs were preincubated for 10 min with inhibitor before addition of NADPH (1 mM). Incubates were extracted, and the CYP-HETEs were purified, derivatized, and quantitated by using GC-MS. Values are means ± SE; n = 4.* P < 0.05, compared with control value.


                              
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Table 1.   Effect of lipase inhibitors on [Sar1]ANG II-stimulated 20-HETE release by rat renal PGMVs

Effect of ANG II infusion on microvascular HETE release. In response to ANG II infusion for 3 days, release from PGMVs increased by twofold, the ratio of 20-HETE to 19-HETE remaining the same (2:1) as observed for acute exposure to ANG II (Fig. 8). No further increase in HETE formation was observed with 14-day ANG II infusion, although blood pressure continued to increase with time; i.e., systolic tail-cuff pressures of unanesthetized control rats and 3- and 14-day ANG II-treated rats were 134 ± 2, 158 ± 6, and 170 ± 15 mmHg, respectively.


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Fig. 8.   In vivo treatment with ANG II (120 ng · kg-1 · min-1) for 3 days increased time-dependent 20-HETE release from rat renal PGMVs. PGMVs were isolated from control or ANG II-treated rats and incubated for 1-20 min. Incubates were extracted, and the CYP-HETEs were purified, derivatized, and quantitated by using GC-MS. Inset: systemic blood pressure (BP; mmHg) of control (134 ± 2 mmHg) and ANG II-treated (158 ± 6 mmHg) rats. Values are means ± SE; n = 4. * P < 0.05 compared with control value.


    DISCUSSION
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ABSTRACT
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The kidney possesses a large capacity to generate CYP-AA products, chiefly the omega - and omega -1 hydroxylase-derived metabolites, 19- and 20-HETEs, and lesser amounts of epoxides, primarily 11,12-EETs (36, 37). However, studies of whole kidney capacity cannot distinguish localization of CYP enzymes in specialized renal tissues, nor can they assign to CYP enzymes their relative importance in the regulation of renal function as, e.g., the high concentrations of omega -hydroxylase in the thick ascending limb (mTAL) and epoxygenase in the medullary collecting ducts (35, 42). The present study identified PGMVs as a potentially important site of interactions involving ANG II and CYP-AA products. There is precedent for ANG II-CYP-AA product interactions in the kidney, having been first described in the nephron. Inhibition of sodium transport by high-dose ANG II in the proximal tubules was dependent on formation of 5,6-EET by this nephron segment (34). In the mTAL, ANG II increased 20-HETE formation, which inhibited the Na+-K+-2Cl- cotransporter by a direct action (14), as well as secondarily, by closing the 70-pS K+ channel, thereby reducing K+ recycling, an essential component in the activity of the cotransporter (29). The effect of ANG II on 5,6-EET formation by the proximal tubules was in response to peptide stimulation of AT2 receptors coupled to PLA2 (18, 26). PLA2 has also been reported to be a target for Ang II in the rat afferent glomerular arteriole, at which site the released AA was metabolized by a lipoxygenase to vasoconstrictor products and by CYP to vasodilator products (22, 23, 31).

The present study is the first to link stimulation of AT2 receptors to production of a vasoconstrictor AA metabolite, 20-HETE, a key component in autoregulation of renal blood flow (54) and tubuloglomerular feedback (55), that acts on the efferent limb of these vascular mechanisms to constrict PGMVs (2, 30). The present study confirms the findings of Imig and colleagues (25) that 20-HETE is a major product of PGMVs and provides definitive identification of 20-HETE formation at this critical site in the renal vasculature on the basis of GC-MS criteria. Under basal conditions, renal PGMVs generated primarily 20-HETE, with lesser amounts of 19-HETE and negligible amounts of 16-, 17-, and 18-HETEs. In response to a 5-min incubation with ANG II, 20-HETE release from PGMVs increased by twofold, a selective effect of ANG II, as neither endothelin-1, phenylephrine, nor bradykinin stimulated release of 20-HETE. ANG II dose dependently stimulated CYP-HETE release; however, the threshold dose varied greatly, e.g., 5-100 nM, presumably related to the level of activity of tissue peptidases that degraded ANG II. A stable analog of ANG II, [Sar1]ANG II, which acts on the same receptor as ANG II, reduced the variability in response to ANG II; [Sar1]ANG II has proven to be a useful agonist for studying activation of phospholipases and release of AA (26). 20-HETE release was increased in a dose-dependent manner by [Sar1]ANG II. Release of preformed 20-HETE from PGMVs by ANG II, as has been reported in the rabbit isolated kidney, was deemed unlikely, as phospholipids isolated from these blood vessels and subject to alkaline hydrolysis did not yield 20-HETE, although moderate quantities of EETs were released.

Although only 5-10% of ANG II receptors are of the AT2 subtype, AT2 receptors are found in the arcuate, interlobular, and afferent arteries in mature human, rabbit, and rat PGMVs (1, 16, 19, 52). Chatziantoniou and Arendshorst (10) have defined two ANG II receptor subtypes in the renal microvasculature of young Wistar-Kyoto and spontaneously hypertensive rats: a classical AT1 receptor accounted for 80% of these sites, whereas 20% displayed a considerably higher affinity for the AT2-receptor antagonist PD-123319 than for losartan. Moreover, both receptor subtypes in the renal microcirculation, those inhibited by either losartan or PD-123319, were functional as they responded to ANG II by vasoconstriction. In the present study, PD- 123319, the AT2 antagonist, blocked release of 19- and 20-HETEs in response to [Sar1]ANG II, whereas the AT1-selective antagonist, losartan, did not; rather, it tended to potentiate the response (Fig. 6), suggesting that the AT1 receptor exerts a countervailing affect on AT2 receptor-mediated stimulation. In segments of rat interlobular arteries, AT2 receptors participate in elevating vascular tone during ANG II-induced constriction, as AT2 receptor blockade partially reversed the vasoconstrictor response to ANG II (19). This finding is in keeping with those of the present study as 20-HETE was released from PGMVs by stimulation of AT2 receptors. Furthermore, 20-HETE has been shown to constrict PGMVs (2, 30). However, in rat afferent arterioles, blockade of AT1 receptors uncovered ANG II-induced dilatation of the afferent arteriole that was abolished by inhibition of epoxygenase activity with miconazole (24), suggesting that activation of AT2 receptors caused dilatation, probably mediated by 11, 12-EET, the most potent EET dilator of the afferent arteriole (23). In contrast, the 5,6-epoxide, usually ranked first of the EETs as a vasodilator epoxide (8), constricted the afferent arteriole (23). Parallel studies in the rabbit by Arima et al. (1) have identified the AT2 receptor of the afferent arteriolar as evoking dilatation when stimulated by ANG II, in the face of AT1 receptor blockade. These studies demonstrating ANG II-induced dilatation of the afferent arteriole, possibly via an AT2 receptor, are in apparent conflict with our findings. However, isolated PGMVs used in the present study are a mixture of interlobar and arcuate arteries and interlobular and afferent arterioles, in contrast to the studies cited that used perfused afferent arterioles attached to glomeruli either with or without attached tubular elements. Furthermore, vascular segments ranging from interlobar arteries to afferent arterioles differ in terms of receptor distribution, reactivity, and localization of oxygenases, the latter generating a different mix of eicosanoids depending on the vascular segment. Moreover, species differences, rabbit vs. rat, can be decisive in determining the final effect of eicosanoid-dependent mechanisms. Therefore, analysis of findings obtained from different renal vascular preparations require cautious interpretation and are rarely, if ever, strictly comparable.

To address the microvascular regulation of ANG II-stimulated HETE formation and release, the lipase responsible for initiating synthesis of 20-HETE by releasing AA substrate to omega -hydroxylase for conversion to 20-HETE was examined. The AT1 receptor engages classic intracellular second messengers; it is G protein coupled to either PLC, PLA2, PLD, or Ca2+ channels, depending on the tissue (48). The signal transduction pathways of the AT2 receptor, on the other hand, are not well understood; they do not appear to be coupled to a G protein (5, 12). In proximal tubules, ANG II was shown to stimulate apical AT2 receptors that activated PLA2 (13, 26). PLA2 stimulation by ANG II has also been demonstrated in the mTAL and in the afferent glomerular arteriole. In the present study, PGMVs were incubated with [Sar1]ANG II in either the presence or absence of PLA2 or PLC inhibitors. Inhibition of either secretory or cytosolic PLA2 did not affect ANG II-induced release of HETEs whereas U-73122, a PLC inhibitor, significantly reduced 20-HETE release produced by ANG II. This is the first report that the AT2 receptor is coupled to PLC and, moreover, is associated with synthesis of a vasoconstrictor product, 20-HETE. This study suggests the potential importance of an AT2-PLC effector unit to constrictor mechanisms in key vascular segments that are primarily responsible for influencing renal vascular resistance.

Finally, as an association between hypertension and increased synthesis of 20-HETE has been reported (41), and as upregulation of AT2 receptors occurred in response to ANG II (15), we infused a pressor dose of ANG II for 2 wk to determine whether 1) renal PGMVs responded to in vivo challenge with ANG II as they did to in vitro exposure to ANG II and 2) ANG II produced sustained elevation in 20-HETE synthesis over a 2-wk period. Microvascular 20-HETE release was increased more than twofold in ANG II-treated groups compared with controls. The enhanced release was evident at 3 days, the earliest time point studied, and was not further enhanced after 14 days of treatment, although blood pressure continued to rise. Thus in vivo ANG II treatment stimulated microvascular 20-HETE production, an effect that was independent of the magnitude of the pressor action of the peptide, at least within the time frame of our study. In contrast to the present study, previous studies in several tissues including skeletal muscle and kidney, have linked AT2 receptors to release of vasodilator products, suggesting that AT2 receptors are related to antihypertensive mechanisms. Indeed, increased expression of renal AT2 receptors has been linked to vasodilator mechanisms in response to decreased dietary salt intake (43). AT2 receptor deletion in knockout mice has been associated with increased blood pressure (20, 21), suggesting that AT2 receptor stimulation moderates/modulates blood pressure-elevating factors by promoting vasodilatation (43, 44). However, stimulation of AT2 receptors in the rat may, under conditions to be determined, elevate blood pressure as they are coupled via PLC to production of the vasoconstrictor eicosanoid, 20-HETE, renal levels of which are increased in spontaneously hypertensive rats during the developmental phase of hypertension (41). In addition, localization of the AT2 receptor-effector complex to the renal microvasculature is important to our understanding of the regulation of the renal circulation as the PGMV is the principal site responsible for producing changes in renal vascular resistance. Elevation of renovascular resistance as an initial event is a sufficient condition for increasing arterial blood pressure. Our findings also call into question general conclusions based on selective disruption of AT2 receptors in mice (20, 21). Although these knockout mice demonstrate hypertension, it may be premature to link AT2 receptors exclusively to antihypertensive mechanisms.


    ACKNOWLEDGEMENTS

We thank Jennifer Brown for secretarial assistance and Melody Steinberg for editorial assistance in preparing this manuscript.


    FOOTNOTES

This research was supported in part by National Heart, Lung, and Blood Institute Grants HL-34300 and HL-25394, and Grant 90-075G from the American Heart Association.

Address for reprint requests and other correspondence: M. A. Carroll, Dept. of Pharmacology, New York Medical College, Valhalla, NY 10595 (E-mail: mairead_carroll{at}nymc.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. §1734 solely to indicate this fact.

Received 4 January 2000; accepted in final form 3 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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