|Home | About | Journals | Submit | Contact Us | Français|
Elevated concentrations of aldosterone are associated with several cardiovascular diseases. Angiotensin 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 cells in the regulation of vascular tone of bovine adrenal cortical arteries by angiotensin II. Zona glomerulosa cells enhance endothelium-dependent relaxations to angiotensin II. The zona glomerulosa cell-dependent relaxations to angiotensin II are unchanged by removing the endothelium-dependent response to angiotensin II. These zona glomerulosa cell-mediated relaxations are ablated by cytochrome P450 inhibition, epoxyeicostrienoic acid antagonism, and potassium channel blockade. Analysis of zona glomerulosa cell epoxyeicosatrienoic acid production by liquid chromatography/mass spectrometry demonstrates an increase in epoxyeicosatrienoic and dihydroxyeicosatrienoic acids with angiotensin II stimulation. These epoxyeicosatrienoic and dihydroxyeicosatrienoic acids produced similar concentration-dependent relaxations of adrenal arteries, which were attenuated by epoxyeicosatrienoic acid antagonism. Whole cell potassium current of adrenal artery smooth muscle cells were increased by angiotensin II stimulation in the presence of zona glomerulosa cells but decreased in the absence of zona glomerulosa cells. This increase in potassium current was abolished by iberiotoxin. Similarly, 14,15-epoxyeicosatrienoic acid induced concentration-dependent increases in potassium current, which was abolished by iberiotoxin. Zona glomerulosa cell aldosterone release is not directly altered by epoxyeicosatrienoic acids. These data suggest that angiotensin II stimulates zona glomerulosa cells to release epoxyeicosatrienoic 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 (CHF) and represents a poor prognosis, presumably due to nonclassical 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 decreases mortality in patients with CHF.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 percent 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 the 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 fasiculata 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 intraadrenal 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 horomone (ACTH).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. ACTH does not affect vascular tone of isolated adrenal cortical arteries in vitro,21 but does induce relaxations in the presence of ZG cells. These ZG cell-mediated relaxations to ACTH are due to the production of cytochrome P450 (CYP450) metabolites of arachidonic acid, namely epoxyeicosatrienoic acids (EETs).22 While 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 demonstrate 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 causes 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 Due to the close association of ZG cells and adrenal cortical arteries and the ability of ZG cells to produce vasoactive factors, the present study will examine whether ZG cells produce vasoactive factors that contribute to the vascular effects of Ang II on adrenal cortical arteries.
Bovine ZG cells and adrenal fibroblasts (AFs) were prepared by enzymatic dissociation of adrenal cortical slices as previously described.28 For vascular reactivity and mass spectrometry 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 (0.01–1 μmol/L) and Ang II (100 nmol/L) for 2 h prior to analysis of media for aldosterone. Aldosterone production by cultured ZG cells was examined by enzyme linked immunosorbant assay as previously described.29
Fresh bovine adrenal glands were acquired from a local slaughterhouse. Subcapsular cortical arteries closely adhered to the adrenal surface (200–300 μm) were dissected and cleaned of connective tissue in ice-cold 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer. Isolated arterial segments were threaded on two 40μm stainless steel wires and mounted in a 610M 4-chamber wire myograph (Danish Myo Technology, Denmark) containing physiological 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, 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 min 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) three times for 10 min at 10 min intervals. Arteries were allowed to equilibrate for 30 min prior to the initiation of experimental protocols.
Arteries were precontracted with submaximal concentrations of U46619 (10–30 nmol/L) to 50–75% 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 if 1 μmol/L acetylcholine caused >90% relaxation and effectively removed if <10% relaxation. Cumulative concentration responses to Ang II (0.1–100 pmol/L) were performed. To examine vasoactive factors released by ZG cells in response to Ang II stimulation, experiments were performed in the presence of ZG cells (5–10 ×105) in intact and denuded arteries pretreated with the endothelial NO synthase inhibitor nitro-L-arginine (L-NA) (30 μmol/L) and the cyclooxygenase (COX) inhibitor indomethacin (10 μmol/L). Responses were repeated with arteries and ZG cells pretreated with the cytochrome P450 (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-, and 14,15-EET and -dihydroxyeicosatetraenoic acids (DHETs) (1 pmol/L-10 μmol/L) were performed in intact vessels in the presence or absence of 14,15-EEZE (10 μmol/L).
ZG cells (5×106) were incubated in 6 mL HEPES buffer with 0.1 or 100 nmol/L Ang II, bubbled with 95% O2, 5% CO2 at 37°C for 10 min. Incubations were stopped by adding ethanol to 15% and buffer and cells were separated by centrifugation. The buffer was removed and extracted using 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 was determined by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS) as previously described.31 Briefly, reconstituted samples were separated on a reverse-phase Kromasil C18 column (250×2 mm) and a Waters 2695 liquid chromatograph (Waters Corporation, 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 min linear gradient from 50% solvent B to 100% solvent B with a flow rate of 0.2 mL/min. Mass spectrometry was performed using a Micromass Quattro Micro API mass spectrometer (Waters Corporation) equipped with an electrospray ionization 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 K+ currents were obtained as previously described.32, 33 Recordings of K+ currents were performed in the presence of 14,15-EET (0.01–1 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.
Ang II (0.1–100 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 stimulates 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 nitric oxide synthase (eNOS) inhibitor L-NA (30 μmol/L), thus eliminating the endothelium-mediated relaxation responses to Ang II pharmacologically. The COX 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 due 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 of the 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 AFs 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 (Figures 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 mediated the relaxations of adrenal arteries in response to Ang II.
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 approximately ten times greater than EETs (Figure 4B). All four DHET regioisomers (5,6-DHET, 8,9-DHET, 11,12-DHET, and 14,15-DHET) were detected and 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.
The three EET regioisomers that were released by ZG cells produced similar concentration-dependent relaxations with maximal relaxation at 10 μmol/L (Figure 5A–C). 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 was observed in the bovine coronary arteries.33 Concentration-dependent relaxations were similar with the three major DHET regioisomers (Figure 5D–F) and correlates well 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 to adrenal arteries or adrenal endothelial cells (Figure S1; please see http://hyper.ahajournals.org). However, the relaxations to 14,15-EET were similar in the presence and absence of ZG cells (Figure 5A and and4C).4C). In the presence of ZG cells, inhibition of sEH by 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 due to the similar activities of 14,15-EET and 14,15-DHET.
Whole cell, outward K+ currents were measured following 10 mV depolarizing steps from -60–60 mV in isolated bovine adrenal cortical artery smooth muscle cells. 14,15-EET (0.01–1 μmol/L) increased outward K+ 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 K+ channels of isolated bovine adrenal cortical artery smooth muscle cells.
ZG cells (1.67 × 105 cells/mL) increase outward K+ 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 increases 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 decreases current density to 70.1%.35
To determine if EETs stimulate steroidogenesis, cultured ZG cells were incubated with 14,15-EET (0.01–1 μmol/L) and aldosterone secretion was measured in the incubation medium. Under basal conditions, ZG cells produced 72.3 pg aldosterone / mL of media (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 allow for paracrine signaling between steroidogenic and vascular cells. The steroidogenic agonist ACTH 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 two 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 vivo,24, 36, 37 and even reduces adrenal blood flow in rats treated with an eNOS inhibitor.38 Thus, endothelial 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. COX 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 artery 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 COX metabolites in the regulation of bovine adrenal vascular tone.22, 30, 40
Using a 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 immnohistochemistry confirm that ZG cells express high levels of sEH. While our previous studies demonstrated that ZG cells produce EETs or related epoxy-metabolites 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, the 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 correlates well with the concentrations of EETs and DHETs required for relaxation. When stimulated with 0.1 nmol/L of Ang II, ZG cells released approximately 2 μmol/L of total EETs and DHETs. Based on the EET and DHET concentration-response curves (Figure 5), this concentration corresponds to 40–50% relaxation and the maximal relaxation observed with Ang II and ZG cells (Figures 1–3).
Whole-cell patch clamp studies demonstrate that co-incubation of ZG cells and Ang II significant increase outward K+ current of adrenal artery smooth muscle cells. In stark contrast, Ang II alone reduces outward K+ current. The increase in smooth muscle cell outward K+ current by Ang II-stimulated ZG cells parallels that of 14,15-EET in magnitude. Similarly, both conditions increase outward K+ 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 using 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, CHF, and some forms of hypertension.7, 41 Despite the importance of aldosterone in the progression of these pathologies, our understanding of the intraadrenal regulation of adrenocortical stereroidogenesis and adrenal blood flow remains poor. While Ang II directly relaxes adrenal cortical arteries,26 this study demonstrates 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 Institute of Health (HL-83297 and GM31278) and the Robert A. Welch Foundation.