Angiotensin II Regulates Adrenal Vascular Tone Through Zona Glomerulosa Cell–Derived EETs and DHETs
Elevated concentrations of aldosterone are associated with several cardiovascular diseases. Angiotensin II (Ang II) increases aldosterone secretion and adrenal blood flow. This concurrent increase in steroidogenesis and adrenal blood flow is not understood. We investigated the role of zona glomerulosa (ZG) cells in the regulation of vascular tone of bovine adrenal cortical arteries by Ang II. ZG cells enhanced endothelium-dependent relaxations to Ang II. The ZG cell–dependent relaxations to Ang II were unchanged by removing the endothelium-dependent response to Ang II. These ZG cell–mediated relaxations were ablated by cytochrome P450 inhibition, epoxyeicosatrienoic acid (EET) antagonism, and potassium channel blockade. Analysis of ZG cell EET production by liquid chromatography/mass spectrometry demonstrated an increase in EETs and dihydroxyeicosatrienoic acids with Ang II stimulation. These EETs and dihydroxyeicosatrienoic acids produced similar concentration-dependent relaxations of adrenal arteries, which were attenuated by EET antagonism. Whole-cell potassium currents of adrenal artery smooth muscle cells were increased by Ang II stimulation in the presence of ZG cells but decreased in the absence of ZG cells. This increase in potassium current was abolished by iberiotoxin. Similarly, 14,15-EET induced concentration-dependent increases in potassium current, which was abolished by iberiotoxin. ZG cell aldosterone release was not directly altered by EETs. These data suggest that Ang II stimulates ZG cells to release EETs and dihydroxyeicosatrienoic acids, resulting in potassium channel activation and relaxation of adrenal arteries. This provides a mechanism by which Ang II concurrently increases adrenal blood flow and steroidogenesis.
The renin-angiotensin-aldosterone system is a major long-term regulator of blood pressure.1 Angiotensin II (Ang II) and aldosterone are the primary effector molecules of the system. Ang II is a potent vasoconstrictor, enhances the activity of the sympathetic nervous system, and stimulates aldosterone secretion.2 Inhibition of Ang II synthesis or Ang II receptor antagonism lowers blood pressure and reduces hypertension.3 Aldosterone is a mineralocorticoid produced by zona glomerulosa (ZG) cells of the adrenal gland and is involved in the control of water and electrolyte balance.4 Elevated circulating concentrations of aldosterone are associated with congestive heart failure and represents a poor prognosis, presumably due to nonclassic actions of aldosterone in the heart and blood vessels that result in oxidative stress, inflammation, and fibrosis.5 Moreover, in clinical trials, a mineralocorticoid receptor antagonist significantly decreased mortality in patients with congestive heart failure.6,7
Regulation of adrenal blood flow is mediated by a complex combination of neural, humoral, and local mediators.8,9 The adrenal gland is a highly vascularized organ that receives a disproportionately high percentage of cardiac output.9,–,11 Several arteries originating from the aorta, renal, and inferior phrenic arteries supply the adrenal glands. After penetrating the adrenal capsule, these arteries closely adhere to the ZG region, running parallel to or within the ZG region. These vessels are the only resistance arteries in the adrenal gland and therefore, control adrenal vascular resistance and blood flow.12 These subcapsular arteries penetrate the gland and form distinct vascular beds for the adrenal cortex and medulla, arteriae cortices, and arteriae medullae, respectively. The arteriae cortices form an anastomotic network within the ZG region before forming a sinusoid network within the zona fasciculata and zona reticularis. This network creates close associations between vascular endothelial cells and adrenocortical cells, allowing for efficient delivery of stimulants, nutrients, cholesterol, and oxygen to steroidogenic cells and the transfer of steroids into the circulation. Within the networks, classic endothelial cell paracrine factors influence steroidogenic cells.9 For example, endothelial cell–derived nitric oxide (NO) inhibits steroidogenesis.13,–,15 Therefore, there exists a complex intra-adrenal regulation of steroidogenesis involving both vascular endothelial cells and adrenal blood flow.
In addition to Ang II, other major regulators of aldosterone secretion are potassium and adrenocorticotropic hormone.16,17 Factors that stimulate aldosterone secretion also increase adrenal blood flow.11,18,19 Early studies were performed either in vivo or in perfused adrenal glands,20 so it was not possible to determine whether the increase in adrenal blood flow was due to a direct action on the vasculature or an indirect action by stimulated release of vasoactive factors from surrounding adrenal tissue. Recent studies have begun to address this question. Adrenocorticotropic hormone does not affect vascular tone of isolated adrenal cortical arteries in vitro,21 but it does induce relaxations in the presence of ZG cells. These ZG cell–mediated relaxations to adrenocorticotropic hormone are due to the production of cytochrome P450 (CYP450) metabolites of arachidonic acid, namely, epoxyeicosatrienoic acids (EETs).22 Whereas vascular endothelial cells release soluble factors that affect steroidogenesis,14,15,23 this novel observation suggests that ZG cells produce vasoactive factors that decrease vascular tone of adrenal cortical arteries and increase adrenal blood flow.
In vivo assessments of the effect of Ang II on adrenal blood flow have demonstrated either no effect on blood flow24 or decreased blood flow at high concentrations.25 Ang II causes a biphasic response in isolated bovine adrenal cortical arteries. At low concentrations, Ang II causes vasodilation by activation of endothelial cell angiotensin type 2 (AT2) receptors and increases in NO production.26 Higher concentrations of Ang II cause vasoconstriction by activation of AT1 receptors.26 Moreover, metabolism of Ang II in bovine adrenal cortical arteries may result in changes in local Ang II concentrations that may alter vascular resistance and adrenal blood flow.27 Owing to the close association of ZG cells and adrenal cortical arteries and the ability of ZG cells to produce vasoactive factors, the present study examined whether ZG cells produce vasoactive factors that contribute to the vascular effects of Ang II on adrenal cortical arteries.
Materials and Methods
Bovine ZG cells and adrenal fibroblasts were prepared by enzymatic dissociation of adrenal cortical slices, as previously described.28 For vascular reactivity and mass spectrometry (MS) studies, freshly isolated ZG cells were used. For studies of aldosterone release, cultured ZG cells were used.29 Cells were incubated with 14,15-EET (0.01 to 1 μmol/L) and Ang II (100 nmol/L) for 2 hours before analysis of the media for aldosterone. Aldosterone production by cultured ZG cells was examined by ELISA, as previously described.29
Isometric Tension Recording
Fresh bovine adrenal glands were acquired from a local slaughterhouse. Subcapsular cortical arteries closely adhered to the adrenal surface (200 to 300 μm) were dissected and cleaned of connective tissue in ice-cold HEPES buffer. Isolated arterial segments were threaded on two 40-μm stainless steel wires and mounted in a 610M 4-chamber wire myograph (Danish Myo Technology, Aarhus, Denmark) containing physiologic saline solution (119 mmol/L NaCl, 24 mmol/L NaHCO3, 4.7 mmol/L KCl, 2.5 mmol/L CaCl2, 1.18 mmol/L KH2PO4, 1.17 mmol/L MgSO4, 0.026 mmol/L EDTA, and 5.5 mmol/L glucose, pH 7.4), bubbled with 95% O2/5% CO2 at 37°C, as previously described.26,27,30 After 30 minutes of equilibration, arteries were gradually stretched to a resting tension of 1 millinewton and stimulated with KCl (60 mmol/L) and the thromboxane A2 mimetic U46619 (100 nmol/L) 3 times for 10 minutes at 10-minute intervals. Arteries were allowed to equilibrate for 30 minutes before initiation of experimental protocols.
Arteries were precontracted with submaximal concentrations of U46619 (10 to 30 nmol/L) to 50% to 75% of their maximal KCl and U46619 stimulation. Where indicated, the endothelium was removed by gently rubbing the arterial intimal surface with a human hair. The endothelium was considered intact when 1 μmol/L acetylcholine caused >90% relaxation and effectively removed when there was <10% relaxation. Cumulative concentration responses to Ang II (0.1 to 100 pmol/L) were measured. To examine vasoactive factors released by ZG cells in response to Ang II stimulation, experiments were performed in the presence of ZG cells (5 to 10×105) in intact and denuded arteries pretreated with the endothelial NO synthase inhibitor nitro-l-arginine (L-NA, 30 μmol/L) and the cyclooxygenase inhibitor indomethacin (10 μmol/L). Responses were repeated with arteries and ZG cells pretreated with the CYP450 inhibitor SKF-525A (10 μmol/L), the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 10 μmol/L), KCl (60 mmol/L), or the large-conductance calcium-activated potassium (BKCa) channel blocker iberiotoxin (100 nmol/L). In a subset of experiments, cumulative concentration responses to 8,9-, 11,12-, 14,15-EET and -dihydroxyeicosatetraenoic acids (DHETs, 1 pmol/L to 10 μmol/L) were performed in intact vessels in the presence or absence of 14,15-EEZE (10 μmol/L).
ZG Cell Arachidonic Acid Metabolites
ZG cells (5×106) were incubated in 6 mL HEPES buffer with 0.1 or 100 nmol/L Ang II and bubbled with 95% O2/5% CO2 at 37°C for 10 minutes. Incubations were stopped by adding ethanol to 25%, and buffer and cells were separated by centrifugation. The buffer was removed and extracted with the use of C18 Bond-Elut solid-phase extraction columns (Varian, Harbor City, CA) as previously described.30 The samples were evaporated to dryness under a stream of N2 and stored at −80°C until analysis.
The chemical identity and quantification of the major arachidonic acid metabolites were determined by liquid chromatography–electrospray ionization MS (LC-ESI/MS) as previously described.31 In brief, reconstituted samples were separated on a reverse-phase Kromasil C18 column (250×2 mm) and a Waters 2695 liquid chromatograph (Waters Corp, Milford, MA). The mobile phase consisted of solvent A, water containing 0.01% glacial acetic acid, and solvent B, acetonitrile containing 0.01% glacial acetic acid. The program was a 40-minute linear gradient from 50% solvent B to 100% solvent B with a flow rate of 0.2 mL/min. MS was performed with a Micromass Quattro Micro API mass spectrometer (Waters Corp) equipped with an ESI source with detection in the negative mode. For quantitative measurements, the m/z=319, 327, 337, and 345 ions were used for EETs, [2H8]EETs, DHETs, and [2H8]DHETs, respectively. The concentrations of these eicosanoids in the samples were calculated by comparing their ratios of the peak areas to the standard curves.
Bovine adrenal cortical artery smooth muscle cells were freshly isolated, and whole-cell recordings of potassium currents were obtained as previously described.32,33 Recordings of potassium currents were performed in the presence of 14,15-EET (0.01 to 1 μmol/L), Ang II (100 pmol/L), ZG cells (1.67×105 cells/mL), and a combination of ZG cells and Ang II. Recordings were repeated in the presence of iberiotoxin.
All data are expressed as mean±SEM. Significant differences between mean values were evaluated by ANOVA, followed by Student-Newman-Keuls multiple-comparison test. Significance was accepted at a value of P<0.05.
ZG Cell–Mediated Vascular Responses to Ang II
Ang II (0.1 to 100 pmol/L) caused concentration-dependent relaxations of intact bovine adrenal arteries, with a maximal response of 28.4±3.4% at 10−10 mol/L (Figure 1). This result is consistent with our previous findings that low concentrations of Ang II stimulate NO production by the activation of endothelial AT2 receptors.26 In the presence of ZG cells, these relaxations were significantly augmented, with a maximal relaxation of 42.1±4.9% at 100 pmol/L (Figure 1).
To solely examine the ZG cell–mediated relaxation response, Ang II concentration responses were performed in intact adrenal arteries in the presence of ZG cells and the endothelial NO synthase (eNOS) inhibitor L-NA (30 μmol/L), thus pharmacologically eliminating the endothelium-mediated relaxation responses to Ang II. The cyclooxygenase inhibitor indomethacin had no effect on ZG cell–mediated relaxations in response to Ang II (data not shown) and was included in all subsequent preparations. In the absence of ZG cells, Ang II caused no vasorelaxation owing to eNOS inhibition (Figure 2A). However, in the presence of ZG cells, Ang II caused concentration-dependent relaxations of adrenal arteries, with a maximal relaxation of 40.8±4.3% at 10−11 mol/L. These relaxations were abolished by SKF-525A, a CYP450 inhibitor that does not inhibit aldosterone synthesis,22 and the EET antagonist 14,15-EEZE (Figure 2B). Moreover, KCl and the BKCa channel blocker iberiotoxin abolished these relaxations (Figure 2C). These data suggest a role for CYP450-derived EETs in ZG cell–mediated relaxations of adrenal arteries in response to Ang II.
To test the possibility that ZG cell–derived arachidonic acid was metabolized to EETs by the vascular endothelium, Ang II concentration responses were performed in endothelium-denuded vessels in the presence of ZG cells. Removal of the endothelium eliminates both the endothelium-mediated relaxation responses to Ang II and the vascular sources of CYP450 epoxygenase and NO synthase. Ang II caused concentration-dependent relaxations in the presence of ZG cells (maximal relaxation of 48.9±6.2% at 10−11 mol/L); however, no relaxations were observed in the presence of adrenal fibroblasts or in the absence of cells (Figure 3A). In the presence of ZG cells, SKF-525A, 14,15-EEZE, KCl, and iberiotoxin abolished Ang II–induced relaxations (Figure 3B and 3C). These results mirror the findings from L-NA–treated intact vessels in the presence of ZG cells and indicate that ZG cell–derived EETs mediate the relaxations of adrenal arteries in response to Ang II.
Ang II–Stimulated Production of EETs and DHETs by ZG Cells
ZG cells were incubated with 0.1 or 100 nmol/L Ang II, and the incubation buffer was analyzed for EETs and DHETs by LC-ESI/MS. ZG cells produced 8,9-EET, 11,12-EET, and 14,15-EET under basal conditions. When stimulated with Ang II, ZG cell synthesis of these EETs significantly increased in a concentration-dependent manner (Figure 4A). 5,6-EET was not detected. DHETs were detected at concentrations ≈10 times greater than EETs (Figure 4B). All 4 DHET regioisomers (5,6-DHET, 8,9-DHET, 11,12-DHET, and 14,15-DHET) were detected and were significantly increased when cells were incubated with Ang II (Figure 4B). These data demonstrate that Ang II stimulates EET and DHET synthesis and release from ZG cells.
Vascular Responses to EETs and DHETs
The 3 EET regioisomers that were released by ZG cells produced similar concentration-dependent relaxations, with maximal relaxation at 10 μmol/L (Figure 5A through 5C). The relaxations to 8,9- and 11,12-EET were attenuated by the EET antagonist 14,15-EEZE. 14,15-EEZE abolished the relaxations to 14,15-EET. Similar inhibition has been observed in bovine coronary arteries.33 Concentration-dependent relaxations were similar with the 3 major DHET regioisomers (Figure 5D through 5F) and are well correlated with DHET relaxations in coronary arteries.34
Soluble epoxide hydrolase (sEH), which converts EETs to DHETs, is present in a greater amount in ZG cells compared with adrenal arteries or adrenal endothelial cells (online-only Figure I in the Data Supplement, available at http://hyper.ahajournals.org). However, the relaxations to 14,15-EET were similar in the presence and absence of ZG cells (Figures 5A and 4C). In the presence of ZG cells, inhibition of sEH by 12-[[(tricyclo[220.127.116.11,7]dec-1-ylamino)carbonyl]amino]-dodecanoic acid (AUDA) did not alter the relaxations to 14,15-EET. Thus, metabolism of 14,15-EET to 14,15-DHET by ZG cell sEH does not affect relaxation, probably owing to the similar activities of 14,15-EET and 14,15-DHET.
Activation of Smooth Muscle Potassium Channel Activity by 14,15-EET and ZG Cells
Whole-cell outward potassium currents were measured during 10-mV depolarizing steps from −60 to 60 mV in isolated bovine adrenal cortical artery smooth muscle cells. 14,15-EET (0.01 to 1 μmol/L) increased outward potassium currents in a concentration-dependent manner, with a maximal increase in current density of 261% at 60 mV with 14,15-EET (1 μmol/L, Figure 6). Addition of iberiotoxin reduced current density to 119% of control current density. These results demonstrate that 14,15-EET activates iberiotoxin-sensitive potassium channels of isolated bovine adrenal cortical artery smooth muscle cells.
ZG cells (1.67×105 cells/mL) increased outward potassium currents of bovine adrenal cortical artery smooth muscle cells by 170% at 60 mV (Figure 7). Ang II (100 pmol/L) stimulation of ZG cells further increased the current density of adrenal smooth muscle cells to 246% of control current density. This increase in current density was abolished by iberiotoxin. Ang II alone decreased current density to 70.1%.35
Aldosterone Secretion by ZG Cells
To determine whether EETs stimulate steroidogenesis, cultured ZG cells were incubated with 14,15-EET (0.01 to 1 μmol/L), and aldosterone secretion was measured in the incubation medium. Under basal conditions, ZG cells produced 72.3 pg aldosterone per mL of medium (Figure 8A). 14,15-EET did not affect aldosterone secretion. When ZG cells were incubated with Ang II (100 nmol/L), aldosterone release increased to 405.6 pg/mL (Figure 8B). This increase with Ang II was not affected by 14,15-EEZE (Figure 8B) or 14,15-EET (10 μmol/L) (data not shown).
The adrenal architecture is important in regulating steroidogenesis and adrenal blood flow. The close association of adrenal resistance vessels with the ZG region allows for paracrine signaling between steroidogenic and vascular cells. The steroidogenic agonist adrenocorticotropic hormone indirectly relaxes adrenal arteries by stimulating ZG cells to release EETs.22 However, this study demonstrates that another adrenal secretagogue, Ang II, relaxes adrenal arteries through 2 distinct mechanisms: directly by endothelial AT2 receptor activation26 of NO synthesis and indirectly by stimulating ZG cell production of EETs and DHETs.
Consistent with our previous findings, Ang II causes concentration-dependent relaxations of intact bovine adrenal cortical arteries.26 In the presence of bovine ZG cells, Ang II–induced relaxations are significantly augmented. Interestingly, Ang II does not significantly affect adrenal blood flow in vivo24,36,37 and even reduces adrenal blood flow in rats treated with an eNOS inhibitor.38 Thus, NO opposes the Ang II vasoconstriction to maintain adrenal blood flow. Our data suggest that the closely associated ZG cells also contribute to the maintenance of adrenal blood flow.
The ZG cell component of the Ang II–induced relaxations was examined by eliminating the endothelial component with either eNOS inhibition or endothelial denudation. In these conditions, ZG cell–mediated relaxations of adrenal arteries in response to Ang II were similar to those observed with intact adrenal arteries and ZG cells. These ZG cell–mediated relaxations were abolished by CYP450 inhibition, EET antagonism, high extracellular potassium, and BKCa channel blockade. Cyclooxygenase inhibition had no effect. These results suggest a role for CYP450 metabolites that act as hyperpolarizing factors by activation of BKCa channels. Furthermore, Ang II–induced relaxations persist in endothelium-denuded adrenal arteries and ZG cells, ruling out the potential release of arachidonic acid from ZG cells that is then metabolized by vascular endothelial CYP450, as is observed between astrocytes and neurons.39 These data indicate that ZG cells produce vasoactive hyperpolarizing factors.22,30 Moreover, these data further demonstrate a minor role for cyclooxygenase metabolites in the regulation of bovine adrenal vascular tone.22,30,40
With use of an LC-ESI/MS assay,31 14,15-EET, 11,12-EET, and 8,9-EET production by ZG cells was significantly increased with Ang II. Similarly, Ang II significantly increased the release of the DHET metabolites of these EETs. Approximately 10 times more DHETs were released than EETs, suggesting an important role for sEH in ZG cell EET metabolism. Western blot and immunohistochemistry confirm that ZG cells express high levels of sEH (see the online-only supplemental Figure at http://hyper.ahajournals.org). Although our previous studies demonstrated that ZG cells produce EETs or related epoxymetabolites of adrenic acid,22,30 this study is the first to quantify the release of ZG cell–derived EETs and DHETs by an endogenous steroidogenic agonist. The primary ZG cell–produced EETs and DHETs induced concentration-dependent relaxations of bovine adrenal arteries, with relatively similar potencies. The EET antagonist 14,15-EEZE completely abolished the relaxations to 14,15-EET and significantly attenuated the relaxations to the other EETs and DHETs. Interestingly, inhibition of EET and DHET relaxations by 14,15-EEZE is similar to the ability of 14,15-EEZE to inhibit ZG cell–mediated relaxations to Ang II. Moreover, the concentration of EETs and DHETs produced by ZG cells incubated with Ang II is well correlated with the concentrations of EETs and DHETs required for relaxation. When stimulated with 0.1 nmol/L Ang II, ZG cells released ≈2 μmol/L of total EETs and DHETs. On the basis of the EET and DHET concentration-response curves (Figure 5), this concentration corresponds to 40% to 50% relaxation and the maximal relaxation observed with Ang II and ZG cells (Figures 1 through 3).
Whole-cell patch-clamp studies demonstrated that coincubation of ZG cells and Ang II significantly increases the outward potassium current of adrenal artery smooth muscle cells. In stark contrast, Ang II alone reduced the outward potassium current. The increase in smooth muscle cell outward potassium current by Ang II–stimulated ZG cells parallels that of 14,15-EET in magnitude. Similarly, both conditions increased outward potassium current by activation of iberiotoxin-sensitive BKCa channels. These data confirm the LC-ESI/MS results that ZG cells produce and secrete EETs that activate smooth muscle BKCa channels.
Finally, 14,15-EET has no steroidogenic effect on ZG cells and plays no role in the stimulation of ZG cell aldosterone secretion by Ang II. These data suggest that ZG cell–derived EETs and DHETs have no direct autocrine role in aldosterone production. Rather, the EETs and DHETs dilate adrenal arteries and regulate adrenal blood flow in conjunction with steroidogenesis. In this regard, the ZG cell–derived EETs and DHETs may indirectly facilitate Ang II–stimulated steroidogenesis by antagonizing Ang II–induced vasoconstriction and ultimately maintaining or increasing adrenal blood flow. Studies with in vivo models are needed to elucidate further the role of EETs and adrenal blood flow on steroidogenesis.
Aldosterone plays a major role in the vascular alterations associated with atherosclerosis, congestive heart failure, and some forms of hypertension.7,41 Despite the importance of aldosterone in the progression of these pathologies, our understanding of the intra-adrenal regulation of adrenocortical steroidogenesis and adrenal blood flow remains poor. Whereas Ang II directly relaxes adrenal cortical arteries,26 this study has demonstrated that Ang II indirectly relaxes adrenal cortical arteries by stimulating the release of EETs and DHETs from ZG cells. This evidence demonstrates that EETs and DHETs are adrenal paracrine mediators whose release is concurrent with steroid production.
Sources of Funding
These studies were supported by grants from the National Institutes of Health (HL-83297 and GM31278), the Robert A. Welch Foundation, and the American Heart Association Midwest Affiliate.
- Received June 21, 2010.
- Revision received July 11, 2010.
- Accepted December 8, 2010.
- © 2011 American Heart Association, Inc.
- Ferrario CM
- Pitt B,
- Reichek N,
- Willenbrock R,
- Zannad F,
- Phillips RA,
- Roniker B,
- Kleiman J,
- Krause S,
- Burns D,
- Williams GH
- Vinson GP,
- Pudney JA,
- Whitehouse BJ
- Hanke CJ,
- Campbell WB
- Hanke CJ,
- O'Brien T,
- Pritchard KA Jr.,
- Campbell WB
- Spat A,
- Hunyady L
- Hinson JP,
- Vinson GP,
- Whitehouse BJ
- Hinson JP,
- Vinson GP,
- Whitehouse BJ,
- Price GM
- Blair-West JR,
- Coghlan JP,
- Denton DA,
- Fei DT,
- Hardy KJ,
- Scoggins BA,
- Wright RD
- Gauthier KM,
- Zhang DX,
- Cui L,
- Nithipatikom K,
- Campbell WB
- Kopf PG,
- Zhang DX,
- Gauthier KM,
- Nithipatikom K,
- Yi XY,
- Falck JR,
- Campbell WB
- Campbell WB,
- Gebremedhin D,
- Pratt PF,
- Harder DR
- Gauthier KM,
- Jagadeesh SG,
- Falck JR,
- Campbell WB
- Campbell WB,
- Deeter C,
- Gauthier KM,
- Ingraham RH,
- Falck JR,
- Li PL
- Lu T,
- Zhang DM,
- Wang XL,
- He T,
- Wang RX,
- Chai Q,
- Katusic ZS,
- Lee HC
- Schiffrin EL