Angiotensin II Dilates Bovine Adrenal Cortical Arterioles: Role of Endothelial Nitric Oxide
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内分泌学杂志 2005年第8期
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
Address all correspondence and requests for reprints to: William B. Campbell, Ph.D., Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226. E-mail: wbcamp@mcw.edu.
Abstract
Adrenal steroidogenesis is modulated by humoral and neuronal factors and blood flow. Angiotensin II (AII) stimulates adrenal cortical aldosterone and cortisol production and medullary catecholamine release. However, AII regulation of adrenal vascular tone has not been characterized. We examined the effect of AII on diameters of cannulated bovine adrenal cortical arteries. Cortical arteries (average internal diameter = 230 μm) were constricted with U46619 and concentration-diameter responses to AII (10–13 to 10–8 mol/liter) were measured. In endothelium-intact arteries, AII induced dilations at low concentrations (maximum dilation = 25 ± 6% at 10–10 mol/liter) and constrictions at high concentrations (maximum constriction = 25 ± 18% at 10–8 mol/liter). AII constrictions were blocked by the angiotensin type 1 (AT1) receptor antagonist, losartan (10–6 mol/liter). AII dilations were enhanced by losartan (maximal dilation = 48 ± 8%), abolished by endothelial cell removal or N-nitro-L-arginine (L-NA, 3 x 10–5 mol/liter) and inhibited by the angiotensin type 2 (AT2) receptor antagonist, PD123319 (10–6 mol/liter, maximal dilation = 18 ± 4%). In a 4,5-diaminofluorescein diacetate nitric oxide (NO) assay of isolated cortical arteries, AII stimulated NO production, which was abolished by PD123319, L-NA, or endothelial cell removal. Western immunoblot of arterial homogenates and endothelial and zona glomerulosa cell lysates revealed 48-kD and 50-kD bands corresponding to AT1 and AT2 receptors, respectively, in all three and a 140-kD band corresponding to endothelial NO synthase in endothelial cells and arteries. Our results demonstrate that AII stimulates adrenal cortical arterial dilation through endothelial cell AT2 receptor activation and NO release and AT1 receptor-dependent constriction.
Introduction
THE ADRENAL GLAND is highly vascularized with arteriolar complexes specific to the cortex and medulla. Capsular and subcapsular arteries branch to form the cortical and medullary arterioles and capillaries, which drain into the central adrenal vein (1, 2, 3). Adrenal blood flow is tightly regulated through numerous neural and humoral influences (3). Even under conditions of hemorrhage and hypotension, adrenal blood flow is maintained (4, 5, 6, 7), although the mechanisms of this regulation are poorly understood. Importantly, increases in adrenal flow alone can stimulate steroid hormone release (8). This implicates a role of adrenal blood flow in the direct regulation of adrenal hormone release and/or production. Agents that stimulate adrenal steroid hormone release may also increase adrenal blood flow (9). Increased adrenal blood flow enhances the rate of stimulant delivery and nutrient and oxygen supplies and assists with hormone release into the systemic circulation. Previous examinations of adrenal flow were performed in vivo or in the perfused adrenal gland. However, in the perfused adrenal gland or the in vivo model, it is not possible to determine whether infused agents alter blood flow through direct interaction with the vasculature or whether the alterations are secondary to the production of vasoactive factors produced and released by the adrenal stromal cells. Furthermore, in whole animal studies, infused vasoactive agents will alter systemic arterial blood pressure, which can also influence adrenal blood flow.
Angiotensin II (AII) is a major regulator of adrenal steroid hormone and catecholamine release (10, 11, 12, 13). Additionally, it is an important regulator of vascular tone and blood pressure (14). The effects of AII on arterial tone vary, depending on concentration, vascular bed, and methodological approach of measurement. AII causes potent vasoconstrictions in numerous arteries, mainly through the activation of AII type 1 receptors (AT1) (14). AII also causes vascular relaxation in some preparations through the activation of AII type 2 receptors (AT2) (15).
Capsular and subcapsular arteries and arterioles are a primary site of adrenal blood flow regulation (1, 7). Small capsular and subcapsular arteries from bovine adrenal glands with diameters of 200–300 μm are the size of resistance arteries (16). Arteries of this size display myogenic pressure-dependent contractile activity, which is an important regulator of tissue perfusion (17). Additionally, small bovine adrenal cortical arteries demonstrate potent contractions to the thromboxane agonist, U46619, 5-hydroxytryptamine, and endothelin-1 as well as endothelium-dependent relaxations to acetylcholine and arachidonic acid (16, 18). Therefore, adrenal cortical arteries display vascular responses consistent with resistance arteries, which advocates for their role in the regulation of adrenal blood flow. However, the effect of AII on adrenal vascular tone is not known.
The purpose of this study was to examine the effects of AII on adrenal vascular tone in isolated small bovine adrenal cortical arteries. The isolated preparation separates the vascular actions of AII from AII-stimulated steroidogenesis or contributions of other adrenal cortical cells. Using this approach, low concentrations of AII caused vasodilation mediated by AT2 receptor activation and endothelial cell nitric oxide (NO) release, whereas higher concentrations induced smooth muscle AT1 receptor-dependent constrictions. These results suggest that AII-stimulated adrenal hormone release may occur through the direct activation of adrenal zona glomerulosa (ZG) cells and secondarily by dilation of adrenal cortical arteries to increase adrenal blood flow.
Materials and Methods
Isolated adrenal cortical artery perfusion
Fresh bovine adrenal glands were obtained from a local abattoir, placed in ice-cold HEPES buffer, and transported immediately to the laboratory. Small subcapsular arteries (170–320 μm) closely attached to the adrenal cortical surface were dissected and cleaned of connective tissues. Arteries were cannulated on glass pipettes in a heated (37 C) lucite perfusion, superfusion chamber and pressurized to 60 mm Hg by a gravity-feed reservoir. The arteries were perfused and superfused with a physiological saline solution (PSS) of the following constituents (in millimoles): 119 NaCl, 4.7 KCl, 1.6 CaCl2, 1.17 MgSO4, 5.5 glucose, 24 NaHCO3, 1.18 NaH2PO4, and 0.026 EDTA. The perfusate and superfusate solutions were equilibrated with 21% O2/5% CO2/74% N2 gas to maintain a pO2 of 140 mm Hg and a pH of 7.4 (19). Digital images of arterial segments were captured and analyzed using a SMZ 1000 zoom stereomicroscope (Nikon Instruments Inc., Melville, NY), Spot Insight camera (Spot Diagnostic Instruments, Sterling Heights, MI) and Dell Pentium computer (Round Rock, TX) with MetaVue software (Universal Imaging Corp., Downingtown, PA). After cannulation, the arteries were equilibrated for a minimum of 30–45 min. Arteries were contracted with a submaximal concentration of U46619 (2 x 10–8 – 2 x 10–7 mol/liter) to achieve approximately 50% constriction from control diameter and vascular responses were recorded. The presence of functional endothelium was determined by dilations to acetylcholine (10–7 mol/liter) (18). In some arteries, the endothelium was removed with an air bolus, (20) and disruption of endothelial function was demonstrated by a lack of acetylcholine dilation. Concentration-dependent changes in arterial diameter were determined for AII (10–13 to 10–8 mol/liter), 14,15-epoxyeicosatrienoic acid (14,15-EET, 10–9 to 10–5 mol/liter), and (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate) (10–8 to 10–4 mol/liter), which were added to the bath solutions. In some experiments, arteries were incubated for 15 min with losartan (10–6 mol/liter), N-nitro-L-arginine, (L-NA, 3 x 10–5 mol/liter), PD123319 (10–6 mol/liter), or saralasin (10–6 mol/liter) in perfusate and superfusate solutions before the addition of U46619. At the end of each experiment, arteries were perfused and superfused with Ca2+-free PSS to determine maximal passive diameter. Diameter changes are expressed as percent dilation relative to U46619 contraction with 100% dilation representing the passive diameter in Ca2+-free PSS.
Measurement of endothelial cell intracellular NO
Small bovine adrenal arteries were cut open along their longitudinal axis and pinned to a Sylgard-coated dish with lumen exposed. After equilibration in PSS for 1 h at 37 C, arteries were incubated at room temperature for 30 min with 4,5-diaminofluorescein diacetate (DAF-2, 10–5 mol/liter), a fluorescent indicator of intracellular NO. DAF-2 fluorescence was monitored at 490 nm excitation and 510–560 nm emission. Fluorescent images were captured, and intensity was analyzed using a PC-controlled charge-coupled device camera and MetaView software as previously described (21). Fluorescence was monitored under control conditions with and without endothelium and after the addition of AII (10–10 mol/liter). In a set of experiments, the arteries were pretreated with PD123319 (10–6 mol/liter). Results are expressed as integrated fluorescence intensity within the area observed, compared with fluorescence under control conditions with endothelium.
Western blot analysis for AT1, AT2, and endothelial NO synthase (eNOS)
Bovine adrenal microvascular endothelial cells and ZG cells were cultured as previously described (22). Freshly isolated bovine cortical adrenal arteries or primary cultured adrenal microvascular endothelial cells and ZG cells were homogenized and proteins subjected to electrophoretic separation. AT1 receptor, AT2 receptor, and eNOS immunoblot analyses were performed as previously described (22, 23). Proteins (10 μg) were loaded onto gels and electrophoretically separated on 10% polyacrylamide Ready Gels (Bio-Rad Laboratories, Hercules, CA) at 150 V for 1 h. Proteins were transferred to nitrocellulose membranes (Bio-Rad) and the membranes were incubated in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% milk at room temperature for 2 h. After washing, membranes were incubated overnight at 4 C with a 1:1000 dilution of rabbit polyclonal anti-AT1 or AT2 receptor antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) or a mouse monoclonal eNOS antibody (1:1000, Transduction Laboratories, Lexington, KY) in TBS blocking solution. Membranes were washed and incubated at room temperature for 1 h with goat antirabbit or rabbit antimouse IgG-horseradish peroxidase-conjugated secondary antibody (1:5000) in TBS containing 5% nonfat dry milk. Immunoreactive bands were detected by a chemiluminescence detection kit (PerkinElmer, Boston, MA) and film (BioMax ML; Eastman Kodak, Rochester, NY).
Materials
Acetylcholine, L-NA, saralasin, and PD123319 were purchased from Sigma (St. Louis, MO). AII was purchased from Peninsula Laboratories Inc (San Carlos, CA). U46199 was purchased from BioMol (Plymouth Meeting, PA). DAF-2 was purchased from Calbiochem (Darmstadt, Germany). DETA NONOate was purchased from Cayman Chemical Co. (Ann Arbor, MI) and losartan was a gift from Merck (Whitehouse Station, NJ). 14,15-EET was synthesized as previously described (24). L-NA was prepared as a 10–1 mol/liter stock in 0.1 N HCl. U46619 was prepared as a 2 x 10–3 mol/liter stock in 95% ethanol, and 14,15-EET was prepared as a 10–2 mol/liter stock in 95% ethanol. All other drugs were prepared to appropriate stock concentrations in distilled water or HEPES buffer.
Statistics
Statistical analysis was performed using ANOVA to determine the significant differences within groups with subsequent Student Neuman Keul’s post hoc analysis used to determine the significance between groups. Data are expressed as mean ± SE of the mean.
Results
Vascular responses to AII
In isolated, perfused bovine adrenal arteries that were preconstricted with U46619, AII (10–13 to 10–8 mol/liter) induced a biphasic diameter response. The images in Fig. 1A show the responses of one artery with an intact endothelium to AII. Resting internal diameter (ID) of this artery of 220 μm decreased to 150 μm with the addition of U46619 (10–8 mol/liter). AII (10–10 mol/liter) increased ID by 25% to 180 μm. The further addition of AII to 10–8 mol/liter reversed the dilation to a constriction (ID = 75 μm). Pretreatment with the AT1 receptor antagonist losartan (10–6 mol/liter) eliminated the constriction to AII. Averaged diameter responses to AII of isolated arteries with an intact endothelium are graphed in Fig. 1B. AII-induced dilations averaged 25 ± 6% at 10–10 mol/liter, and constrictions averaged 25 ± 18% at 10–8 mol/liter. Treatment of the arteries with losartan abolished the AII-induced constrictions, and AII caused only dilations at all concentrations tested. Losartan also enhanced the AII-induced dilations. Maximal dilations in the presence of losartan averaged 48 ± 8% at 10–8 mol/liter AII. In endothelium-denuded arteries, only AII-induced constrictions were observed (Fig. 1C). Maximal constrictions averaged 44 ± 5% at 10–8 mol/liter AII. This constrictor response to AII was eliminated by pretreatment with losartan. These results demonstrate that AII induces endothelium-dependent dilations at low concentrations, which reverses to a smooth muscle, AT1-dependent constriction at higher concentrations. Furthermore, the AII dilations are modulated by AT1-dependent constrictions, and the constrictions are modulated by endothelial cell, AT2-dependent dilations.
FIG. 1. Effects of AII on the diameter of small perfused bovine cortical adrenal arteries. A, Images of an adrenal artery preconstricted with U46619 (control) plus AII (10–10 and 10–8 mol/liter) and losartan (10–6 mol/liter) plus AII (10–8 mol/liter). Effect of losartan on AII-induced dilations in endothelium-intact (B) or endothelium-denuded arteries (C). *, P 0.05 vs. control, n = 5–6.
Effect of NO synthase inhibition on AII-induced dilations
To determine the endothelial cell factor(s) that mediate the AII-induced dilation, we evaluated AII-diameter responses of endothelium-intact arteries in the presence of losartan to enhance the dilation response (Fig. 2A). AII induced maximal dilations that averaged 37 ± 7% at 10–8 mol/liter AII. The dilations were abolished by pretreatment of the arteries with the NO synthase inhibitor, L-NA (3 x 10–5 mol/liter). These results suggest that AII-induced relaxations of bovine adrenal arteries are mediated by endothelial NO.
FIG. 2. Concentration-dependent dilations to AII, DETA NONOate, and 14,15-EET in small bovine cortical adrenal arteries. A, Dilation responses to AII in the presence of losartan (10–6 mol/liter) with and without L-NA (3 x 10–5 mol/liter). Dilation responses to DETA NONOate (B) and 14,15-EET (C) with and without losartan. *, P 0.05 vs. losartan or control, n = 3–5.
Effect of losartan on NO and 14,15-EET dilations
To demonstrate that losartan did not alter the ability of the arteries to relax to endothelium-independent dilator agonists, we examined diameter responses to the NO donor, DETA NONOate, and 14,15-EET in endothelium-denuded arteries (Fig. 2, B and C). DETA NONOate and 14,15-EET caused concentration-related dilations of the adrenal cortical arteries. Preincubation with losartan did not alter the dilations to the NO donor or 14,15-EET. These results demonstrate that losartan does not alter the dilation response of isolated adrenal arteries.
AII-stimulated NO production measured by DAF-2 fluorescence
To further investigate AII-induced NO synthesis in bovine adrenal cortical arteries, we examined NO production using a DAF-2 fluorescence assay (21). Figure 3A shows images of DAF-2 fluorescence of the endothelial surface of a small cortical artery. Under control conditions minimal fluorescence was observed. The addition of AII (10–10 mol/liter) stimulated an increase in fluorescence. Pretreatment with L-NA (3 x 10–5 mol/liter) or endothelium removal abolished the AII-stimulated fluorescence. Relative fluorescence responses were determined and compared with fluorescence observed in control conditions (Fig. 3B). AII increased fluorescence intensity more than 3-fold, compared with control values, in endothelium-intact arteries. The AII-induced fluorescence was blocked by pretreatment with L-NA, PD123319 (10–6 mol/liter), or endothelial cell removal. These results confirm that AII stimulates endothelial NO production of adrenal cortical arteries.
FIG. 3. Effect of AII on NO-dependent DAF-2 fluorescence from luminal surfaces of small bovine cortical adrenal arteries. Images of DAF-2 fluorescence in arteries with endothelium under control conditions, AII (10–10 mol/liter), L-NA (3 x 10–5 mol/liter) plus AII or after endothelial cell removal (EC–) plus AII (A). Averaged DAF-2 fluorescent intensity under control conditions, AII (10–10 mol/liter), PD123319 (PD, 10–6 mol/liter), PD123319 and AII, L-NA and AII or after endothelial cell removal (EC–) plus AII (B). *, P 0.05 vs. control; #, P 0.05 vs. AII, n = 3–6.
Effect of PD123319 and saralasin on AII-induced dilations
To determine the AII receptor(s) that initiate endothelial cell NO production, we evaluated the effect of the AT2 receptor antagonist, PD123319, on AII-induced dilations of adrenal cortical arteries in the presence of losartan (10–6 mol/liter, Fig. 4). Maximal AII-induced dilations averaged 32 ± 6% at 10–8 mol/liter AII. Pretreatment with PD123319 (10–6 mol/liter) reduced AII-induced dilations by 61% (maximal dilations at 10–8 mol/liter AII = 12.5 ± 6%). Similarly, pretreatment with the nonspecific AII receptor antagonist, saralasin (10–6 mol/liter), reduced AII-induced dilations by 72% at 10–8 mol/liter AII (maximal dilations = 9 ± 9%). These results suggest that AII-induced endothelial NO release and the subsequent dilations are mediated by AT2 receptor stimulation.
FIG. 4. Effect of the AT receptor antagonists, PD123319 (PD, 10–6 mol/liter) and saralasin (10–6 mol/liter), on AII-induced dilations of small bovine cortical adrenal arteries treated with losartan (10–6 mol/liter). *, P 0.05 vs. losartan, n = 5, each.
Western immunoblot of AT receptors and eNOS protein expression
Western immunoblot analysis of proteins isolated from bovine adrenal arteries, adrenal microvascular endothelial cells, and ZG cells revealed expression of AT1 and AT2 receptors (Fig. 5). Immunoreactive bands of 48 and 50 kDa corresponding to AT1 and AT2 receptors, respectively, were observed with adrenal arteries, adrenal endothelial cells, and ZG cells. A 140-kDa immunoreactive band corresponding to eNOS was also observed with adrenal arteries and adrenal endothelial cells but not ZG cells. This demonstrates that adrenal cortical arteries express AT receptors that mediate the AII vasoconstriction (AT1) and vasodilation (AT2) as well as endothelial cell NO synthase.
FIG. 5. Western immunoblot analysis of AT receptors and eNOS expression in bovine adrenal arteries (BAA), bovine adrenal microvascular endothelial cells (BMEC), and bovine ZG cells.
Discussion
This is the first study to examine the effects of AII on the diameters of isolated small adrenal cortical arteries. AII caused a biphasic response. At low concentrations, vasodilation was observed that reversed to a constriction at higher concentrations. Previously we had demonstrated that bovine adrenal cortical arterioles also constrict to the thromboxane analog U46619, serotonin, and endothelin-1 (16). The AII-induced constriction was mediated by the AT1 receptor subtype. This was indicated by blockade of the AII-induced constriction with the AT1 receptor antagonist losartan and verification of AT1 receptor expression in the cortical arteries. It is well established that AII-dependent constrictions in arteries from numerous vascular beds are mediated by the AT1 receptor subtype (14, 25). The presence of losartan enhanced the dilations to AII. Conversely, endothelial cell removal enhanced the constrictions to AII. This suggests that AII-induced constrictions of adrenal cortical arteries are modulated by relaxing factors produced by the endothelium.
Vasodilation by AII was blocked by endothelial cell removal and the NO synthase inhibitor, L-NA, and partially inhibited by the AT2 receptor antagonist, PD123319. Further inhibition of the AII-dependent relaxations with the nonspecific AII antagonist, saralasin, was not observed. These data suggest that AII vasodilation of the cortical arteries is mediated to a large extent by activation of endothelial cell AT2 receptors, resulting in NO production. Using the DAF-2 assay, we demonstrated that AII stimulated NO-dependent fluorescence in the endothelium of the cortical arteries. The AII-induced increase in fluorescence was also blocked by PD123319. AT2 receptor and eNOS protein expression were verified in adrenal endothelial cells and adrenal cortical arteries by Western immunoblot. AT2-dependent vasodilations and relaxations to AII have been observed in numerous arteries including renal, cerebral, mesenteric, and coronary arteries (26, 27, 28, 29, 30). Similar to the adrenal cortical arteries, AII relaxations in coronary arteries were mediated by NO (30). In the kidney, NO is believed to mediate AT2 receptor function (31). Therefore, contribution of NO to the AT2-dependent dilations of the perfused cortical arteries is consistent with results in other vascular beds.
In the adrenal cortical arteries, residual dilations remained after inhibition of AT2 receptors with PD123319. The residual dilations could be due to incomplete blockade of AT2 receptors or the activation of other AII receptor subtypes on the adrenal endothelium not sensitive to inhibition by PD123319 or losartan. In this regard, AT4 receptors have been identified in bovine vascular endothelial cells and bovine adrenal glands (32, 33). However, the role of AT4 receptors in mediating endothelium-dependent dilations to AII are not clear. Additionally, AII vascular activity has been linked to transactivation of receptor tyrosine kinases (34, 35) and a role of receptor tyrosine kinases in the NO-dependent dilations to AII should be considered.
A biphasic response to AII is not unique to the adrenal circulation. In perfused porcine coronary arteries, AT1-dependent constriction to AII was observed at low concentrations and reversed to a AT2-dependent dilation with higher concentrations (29). In the mesenteric vascular bed of the rat, infusion of AII induced a vasodilation followed by a constriction (36). Additionally, AT2-dependent relaxations are often revealed only after selective antagonism of the AT1 receptor (27, 30). Thus, in several vascular beds, AII regulation of vascular tone is complex and involves both AT1 and AT2 receptor subtype activation.
In addition to direct vasodilation and the modulation of AII constriction, the AII-induced NO release could inhibit the secretion of aldosterone. NO binds to cytochrome p450 in adrenal cells and inhibits steroidogenesis (22). Endothelial cell NO partially inhibits AII-stimulation of aldosterone release from cultured bovine ZG cells (22). Because ZG cells are in close proximity to the capsular and subcapsular arterioles, it is possible that AII-stimulated endothelial cell NO acts as a negative paracrine regulator of aldosterone secretion.
Previous in vivo studies showed that AII infusion had either no effect or decreased adrenal blood flow. In sheep with cervical adrenal autotransplants or dexamethasone-treated rabbits, AII infusion did not alter adrenal blood (37, 38, 39, 40, 41). In rats infusions of high concentrations of AII decreased adrenal blood flow (42, 43), which was further reduced by the NO synthase inhibitor, N-omega-nitro-L-arginine methyl ester (43). Thus, it appears AII-induced alterations of adrenal blood flow in vivo are modulated by NO. These findings are in agreement with our findings in the isolated cortical arteries. However, in the in vivo model, the vasodilatory effect of AII and the resulting increase in adrenal blood flow are masked by other physiological effects of the peptide. Because adrenal cortical arteries are a site of myogenic regulation (7), the increase in systemic blood pressure by AII would result in pressure-induced constriction of the adrenal cortical arteries. This constriction could supersede the direct vasodilatory effects of AII.
In the human, normal plasma concentrations of AII are 9–36 pg/ml (8–30 pM) (44, 45, 46). Correspondingly, AII plasma concentrations in unrestrained bovine steers average 55–65 pM (47, 48). These concentrations correlate to a vasodilator effect in adrenal cortical arteries of this study. Increasing circulating AII concentrations 4- to 15-fold above basal values would further dilate the cortical arteries and increase adrenal blood flow. A concentration of 120 pg/ml (10 nM) corresponds to the maximal AII dilatory state of bovine adrenal cortical arteries. Further increases in AII concentrations would stimulate a constrictor response and result in decreased adrenal blood flow. In a small number of active, upright, furosemide-treated hypertensive patients, without evidence of primary aldosteronism, AII plasma concentrations in excess of 200 pg/ml have been measured (44). Thus, high pathophysiological levels of AII would stimulate constriction of adrenal cortical arteries that would limit AII delivery to the adrenal gland and secretion of aldosterone to the circulation.
Acknowledgments
We thank Gretchen Barg for secretarial assistance.
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Address all correspondence and requests for reprints to: William B. Campbell, Ph.D., Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226. E-mail: wbcamp@mcw.edu.
Abstract
Adrenal steroidogenesis is modulated by humoral and neuronal factors and blood flow. Angiotensin II (AII) stimulates adrenal cortical aldosterone and cortisol production and medullary catecholamine release. However, AII regulation of adrenal vascular tone has not been characterized. We examined the effect of AII on diameters of cannulated bovine adrenal cortical arteries. Cortical arteries (average internal diameter = 230 μm) were constricted with U46619 and concentration-diameter responses to AII (10–13 to 10–8 mol/liter) were measured. In endothelium-intact arteries, AII induced dilations at low concentrations (maximum dilation = 25 ± 6% at 10–10 mol/liter) and constrictions at high concentrations (maximum constriction = 25 ± 18% at 10–8 mol/liter). AII constrictions were blocked by the angiotensin type 1 (AT1) receptor antagonist, losartan (10–6 mol/liter). AII dilations were enhanced by losartan (maximal dilation = 48 ± 8%), abolished by endothelial cell removal or N-nitro-L-arginine (L-NA, 3 x 10–5 mol/liter) and inhibited by the angiotensin type 2 (AT2) receptor antagonist, PD123319 (10–6 mol/liter, maximal dilation = 18 ± 4%). In a 4,5-diaminofluorescein diacetate nitric oxide (NO) assay of isolated cortical arteries, AII stimulated NO production, which was abolished by PD123319, L-NA, or endothelial cell removal. Western immunoblot of arterial homogenates and endothelial and zona glomerulosa cell lysates revealed 48-kD and 50-kD bands corresponding to AT1 and AT2 receptors, respectively, in all three and a 140-kD band corresponding to endothelial NO synthase in endothelial cells and arteries. Our results demonstrate that AII stimulates adrenal cortical arterial dilation through endothelial cell AT2 receptor activation and NO release and AT1 receptor-dependent constriction.
Introduction
THE ADRENAL GLAND is highly vascularized with arteriolar complexes specific to the cortex and medulla. Capsular and subcapsular arteries branch to form the cortical and medullary arterioles and capillaries, which drain into the central adrenal vein (1, 2, 3). Adrenal blood flow is tightly regulated through numerous neural and humoral influences (3). Even under conditions of hemorrhage and hypotension, adrenal blood flow is maintained (4, 5, 6, 7), although the mechanisms of this regulation are poorly understood. Importantly, increases in adrenal flow alone can stimulate steroid hormone release (8). This implicates a role of adrenal blood flow in the direct regulation of adrenal hormone release and/or production. Agents that stimulate adrenal steroid hormone release may also increase adrenal blood flow (9). Increased adrenal blood flow enhances the rate of stimulant delivery and nutrient and oxygen supplies and assists with hormone release into the systemic circulation. Previous examinations of adrenal flow were performed in vivo or in the perfused adrenal gland. However, in the perfused adrenal gland or the in vivo model, it is not possible to determine whether infused agents alter blood flow through direct interaction with the vasculature or whether the alterations are secondary to the production of vasoactive factors produced and released by the adrenal stromal cells. Furthermore, in whole animal studies, infused vasoactive agents will alter systemic arterial blood pressure, which can also influence adrenal blood flow.
Angiotensin II (AII) is a major regulator of adrenal steroid hormone and catecholamine release (10, 11, 12, 13). Additionally, it is an important regulator of vascular tone and blood pressure (14). The effects of AII on arterial tone vary, depending on concentration, vascular bed, and methodological approach of measurement. AII causes potent vasoconstrictions in numerous arteries, mainly through the activation of AII type 1 receptors (AT1) (14). AII also causes vascular relaxation in some preparations through the activation of AII type 2 receptors (AT2) (15).
Capsular and subcapsular arteries and arterioles are a primary site of adrenal blood flow regulation (1, 7). Small capsular and subcapsular arteries from bovine adrenal glands with diameters of 200–300 μm are the size of resistance arteries (16). Arteries of this size display myogenic pressure-dependent contractile activity, which is an important regulator of tissue perfusion (17). Additionally, small bovine adrenal cortical arteries demonstrate potent contractions to the thromboxane agonist, U46619, 5-hydroxytryptamine, and endothelin-1 as well as endothelium-dependent relaxations to acetylcholine and arachidonic acid (16, 18). Therefore, adrenal cortical arteries display vascular responses consistent with resistance arteries, which advocates for their role in the regulation of adrenal blood flow. However, the effect of AII on adrenal vascular tone is not known.
The purpose of this study was to examine the effects of AII on adrenal vascular tone in isolated small bovine adrenal cortical arteries. The isolated preparation separates the vascular actions of AII from AII-stimulated steroidogenesis or contributions of other adrenal cortical cells. Using this approach, low concentrations of AII caused vasodilation mediated by AT2 receptor activation and endothelial cell nitric oxide (NO) release, whereas higher concentrations induced smooth muscle AT1 receptor-dependent constrictions. These results suggest that AII-stimulated adrenal hormone release may occur through the direct activation of adrenal zona glomerulosa (ZG) cells and secondarily by dilation of adrenal cortical arteries to increase adrenal blood flow.
Materials and Methods
Isolated adrenal cortical artery perfusion
Fresh bovine adrenal glands were obtained from a local abattoir, placed in ice-cold HEPES buffer, and transported immediately to the laboratory. Small subcapsular arteries (170–320 μm) closely attached to the adrenal cortical surface were dissected and cleaned of connective tissues. Arteries were cannulated on glass pipettes in a heated (37 C) lucite perfusion, superfusion chamber and pressurized to 60 mm Hg by a gravity-feed reservoir. The arteries were perfused and superfused with a physiological saline solution (PSS) of the following constituents (in millimoles): 119 NaCl, 4.7 KCl, 1.6 CaCl2, 1.17 MgSO4, 5.5 glucose, 24 NaHCO3, 1.18 NaH2PO4, and 0.026 EDTA. The perfusate and superfusate solutions were equilibrated with 21% O2/5% CO2/74% N2 gas to maintain a pO2 of 140 mm Hg and a pH of 7.4 (19). Digital images of arterial segments were captured and analyzed using a SMZ 1000 zoom stereomicroscope (Nikon Instruments Inc., Melville, NY), Spot Insight camera (Spot Diagnostic Instruments, Sterling Heights, MI) and Dell Pentium computer (Round Rock, TX) with MetaVue software (Universal Imaging Corp., Downingtown, PA). After cannulation, the arteries were equilibrated for a minimum of 30–45 min. Arteries were contracted with a submaximal concentration of U46619 (2 x 10–8 – 2 x 10–7 mol/liter) to achieve approximately 50% constriction from control diameter and vascular responses were recorded. The presence of functional endothelium was determined by dilations to acetylcholine (10–7 mol/liter) (18). In some arteries, the endothelium was removed with an air bolus, (20) and disruption of endothelial function was demonstrated by a lack of acetylcholine dilation. Concentration-dependent changes in arterial diameter were determined for AII (10–13 to 10–8 mol/liter), 14,15-epoxyeicosatrienoic acid (14,15-EET, 10–9 to 10–5 mol/liter), and (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate) (10–8 to 10–4 mol/liter), which were added to the bath solutions. In some experiments, arteries were incubated for 15 min with losartan (10–6 mol/liter), N-nitro-L-arginine, (L-NA, 3 x 10–5 mol/liter), PD123319 (10–6 mol/liter), or saralasin (10–6 mol/liter) in perfusate and superfusate solutions before the addition of U46619. At the end of each experiment, arteries were perfused and superfused with Ca2+-free PSS to determine maximal passive diameter. Diameter changes are expressed as percent dilation relative to U46619 contraction with 100% dilation representing the passive diameter in Ca2+-free PSS.
Measurement of endothelial cell intracellular NO
Small bovine adrenal arteries were cut open along their longitudinal axis and pinned to a Sylgard-coated dish with lumen exposed. After equilibration in PSS for 1 h at 37 C, arteries were incubated at room temperature for 30 min with 4,5-diaminofluorescein diacetate (DAF-2, 10–5 mol/liter), a fluorescent indicator of intracellular NO. DAF-2 fluorescence was monitored at 490 nm excitation and 510–560 nm emission. Fluorescent images were captured, and intensity was analyzed using a PC-controlled charge-coupled device camera and MetaView software as previously described (21). Fluorescence was monitored under control conditions with and without endothelium and after the addition of AII (10–10 mol/liter). In a set of experiments, the arteries were pretreated with PD123319 (10–6 mol/liter). Results are expressed as integrated fluorescence intensity within the area observed, compared with fluorescence under control conditions with endothelium.
Western blot analysis for AT1, AT2, and endothelial NO synthase (eNOS)
Bovine adrenal microvascular endothelial cells and ZG cells were cultured as previously described (22). Freshly isolated bovine cortical adrenal arteries or primary cultured adrenal microvascular endothelial cells and ZG cells were homogenized and proteins subjected to electrophoretic separation. AT1 receptor, AT2 receptor, and eNOS immunoblot analyses were performed as previously described (22, 23). Proteins (10 μg) were loaded onto gels and electrophoretically separated on 10% polyacrylamide Ready Gels (Bio-Rad Laboratories, Hercules, CA) at 150 V for 1 h. Proteins were transferred to nitrocellulose membranes (Bio-Rad) and the membranes were incubated in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% milk at room temperature for 2 h. After washing, membranes were incubated overnight at 4 C with a 1:1000 dilution of rabbit polyclonal anti-AT1 or AT2 receptor antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) or a mouse monoclonal eNOS antibody (1:1000, Transduction Laboratories, Lexington, KY) in TBS blocking solution. Membranes were washed and incubated at room temperature for 1 h with goat antirabbit or rabbit antimouse IgG-horseradish peroxidase-conjugated secondary antibody (1:5000) in TBS containing 5% nonfat dry milk. Immunoreactive bands were detected by a chemiluminescence detection kit (PerkinElmer, Boston, MA) and film (BioMax ML; Eastman Kodak, Rochester, NY).
Materials
Acetylcholine, L-NA, saralasin, and PD123319 were purchased from Sigma (St. Louis, MO). AII was purchased from Peninsula Laboratories Inc (San Carlos, CA). U46199 was purchased from BioMol (Plymouth Meeting, PA). DAF-2 was purchased from Calbiochem (Darmstadt, Germany). DETA NONOate was purchased from Cayman Chemical Co. (Ann Arbor, MI) and losartan was a gift from Merck (Whitehouse Station, NJ). 14,15-EET was synthesized as previously described (24). L-NA was prepared as a 10–1 mol/liter stock in 0.1 N HCl. U46619 was prepared as a 2 x 10–3 mol/liter stock in 95% ethanol, and 14,15-EET was prepared as a 10–2 mol/liter stock in 95% ethanol. All other drugs were prepared to appropriate stock concentrations in distilled water or HEPES buffer.
Statistics
Statistical analysis was performed using ANOVA to determine the significant differences within groups with subsequent Student Neuman Keul’s post hoc analysis used to determine the significance between groups. Data are expressed as mean ± SE of the mean.
Results
Vascular responses to AII
In isolated, perfused bovine adrenal arteries that were preconstricted with U46619, AII (10–13 to 10–8 mol/liter) induced a biphasic diameter response. The images in Fig. 1A show the responses of one artery with an intact endothelium to AII. Resting internal diameter (ID) of this artery of 220 μm decreased to 150 μm with the addition of U46619 (10–8 mol/liter). AII (10–10 mol/liter) increased ID by 25% to 180 μm. The further addition of AII to 10–8 mol/liter reversed the dilation to a constriction (ID = 75 μm). Pretreatment with the AT1 receptor antagonist losartan (10–6 mol/liter) eliminated the constriction to AII. Averaged diameter responses to AII of isolated arteries with an intact endothelium are graphed in Fig. 1B. AII-induced dilations averaged 25 ± 6% at 10–10 mol/liter, and constrictions averaged 25 ± 18% at 10–8 mol/liter. Treatment of the arteries with losartan abolished the AII-induced constrictions, and AII caused only dilations at all concentrations tested. Losartan also enhanced the AII-induced dilations. Maximal dilations in the presence of losartan averaged 48 ± 8% at 10–8 mol/liter AII. In endothelium-denuded arteries, only AII-induced constrictions were observed (Fig. 1C). Maximal constrictions averaged 44 ± 5% at 10–8 mol/liter AII. This constrictor response to AII was eliminated by pretreatment with losartan. These results demonstrate that AII induces endothelium-dependent dilations at low concentrations, which reverses to a smooth muscle, AT1-dependent constriction at higher concentrations. Furthermore, the AII dilations are modulated by AT1-dependent constrictions, and the constrictions are modulated by endothelial cell, AT2-dependent dilations.
FIG. 1. Effects of AII on the diameter of small perfused bovine cortical adrenal arteries. A, Images of an adrenal artery preconstricted with U46619 (control) plus AII (10–10 and 10–8 mol/liter) and losartan (10–6 mol/liter) plus AII (10–8 mol/liter). Effect of losartan on AII-induced dilations in endothelium-intact (B) or endothelium-denuded arteries (C). *, P 0.05 vs. control, n = 5–6.
Effect of NO synthase inhibition on AII-induced dilations
To determine the endothelial cell factor(s) that mediate the AII-induced dilation, we evaluated AII-diameter responses of endothelium-intact arteries in the presence of losartan to enhance the dilation response (Fig. 2A). AII induced maximal dilations that averaged 37 ± 7% at 10–8 mol/liter AII. The dilations were abolished by pretreatment of the arteries with the NO synthase inhibitor, L-NA (3 x 10–5 mol/liter). These results suggest that AII-induced relaxations of bovine adrenal arteries are mediated by endothelial NO.
FIG. 2. Concentration-dependent dilations to AII, DETA NONOate, and 14,15-EET in small bovine cortical adrenal arteries. A, Dilation responses to AII in the presence of losartan (10–6 mol/liter) with and without L-NA (3 x 10–5 mol/liter). Dilation responses to DETA NONOate (B) and 14,15-EET (C) with and without losartan. *, P 0.05 vs. losartan or control, n = 3–5.
Effect of losartan on NO and 14,15-EET dilations
To demonstrate that losartan did not alter the ability of the arteries to relax to endothelium-independent dilator agonists, we examined diameter responses to the NO donor, DETA NONOate, and 14,15-EET in endothelium-denuded arteries (Fig. 2, B and C). DETA NONOate and 14,15-EET caused concentration-related dilations of the adrenal cortical arteries. Preincubation with losartan did not alter the dilations to the NO donor or 14,15-EET. These results demonstrate that losartan does not alter the dilation response of isolated adrenal arteries.
AII-stimulated NO production measured by DAF-2 fluorescence
To further investigate AII-induced NO synthesis in bovine adrenal cortical arteries, we examined NO production using a DAF-2 fluorescence assay (21). Figure 3A shows images of DAF-2 fluorescence of the endothelial surface of a small cortical artery. Under control conditions minimal fluorescence was observed. The addition of AII (10–10 mol/liter) stimulated an increase in fluorescence. Pretreatment with L-NA (3 x 10–5 mol/liter) or endothelium removal abolished the AII-stimulated fluorescence. Relative fluorescence responses were determined and compared with fluorescence observed in control conditions (Fig. 3B). AII increased fluorescence intensity more than 3-fold, compared with control values, in endothelium-intact arteries. The AII-induced fluorescence was blocked by pretreatment with L-NA, PD123319 (10–6 mol/liter), or endothelial cell removal. These results confirm that AII stimulates endothelial NO production of adrenal cortical arteries.
FIG. 3. Effect of AII on NO-dependent DAF-2 fluorescence from luminal surfaces of small bovine cortical adrenal arteries. Images of DAF-2 fluorescence in arteries with endothelium under control conditions, AII (10–10 mol/liter), L-NA (3 x 10–5 mol/liter) plus AII or after endothelial cell removal (EC–) plus AII (A). Averaged DAF-2 fluorescent intensity under control conditions, AII (10–10 mol/liter), PD123319 (PD, 10–6 mol/liter), PD123319 and AII, L-NA and AII or after endothelial cell removal (EC–) plus AII (B). *, P 0.05 vs. control; #, P 0.05 vs. AII, n = 3–6.
Effect of PD123319 and saralasin on AII-induced dilations
To determine the AII receptor(s) that initiate endothelial cell NO production, we evaluated the effect of the AT2 receptor antagonist, PD123319, on AII-induced dilations of adrenal cortical arteries in the presence of losartan (10–6 mol/liter, Fig. 4). Maximal AII-induced dilations averaged 32 ± 6% at 10–8 mol/liter AII. Pretreatment with PD123319 (10–6 mol/liter) reduced AII-induced dilations by 61% (maximal dilations at 10–8 mol/liter AII = 12.5 ± 6%). Similarly, pretreatment with the nonspecific AII receptor antagonist, saralasin (10–6 mol/liter), reduced AII-induced dilations by 72% at 10–8 mol/liter AII (maximal dilations = 9 ± 9%). These results suggest that AII-induced endothelial NO release and the subsequent dilations are mediated by AT2 receptor stimulation.
FIG. 4. Effect of the AT receptor antagonists, PD123319 (PD, 10–6 mol/liter) and saralasin (10–6 mol/liter), on AII-induced dilations of small bovine cortical adrenal arteries treated with losartan (10–6 mol/liter). *, P 0.05 vs. losartan, n = 5, each.
Western immunoblot of AT receptors and eNOS protein expression
Western immunoblot analysis of proteins isolated from bovine adrenal arteries, adrenal microvascular endothelial cells, and ZG cells revealed expression of AT1 and AT2 receptors (Fig. 5). Immunoreactive bands of 48 and 50 kDa corresponding to AT1 and AT2 receptors, respectively, were observed with adrenal arteries, adrenal endothelial cells, and ZG cells. A 140-kDa immunoreactive band corresponding to eNOS was also observed with adrenal arteries and adrenal endothelial cells but not ZG cells. This demonstrates that adrenal cortical arteries express AT receptors that mediate the AII vasoconstriction (AT1) and vasodilation (AT2) as well as endothelial cell NO synthase.
FIG. 5. Western immunoblot analysis of AT receptors and eNOS expression in bovine adrenal arteries (BAA), bovine adrenal microvascular endothelial cells (BMEC), and bovine ZG cells.
Discussion
This is the first study to examine the effects of AII on the diameters of isolated small adrenal cortical arteries. AII caused a biphasic response. At low concentrations, vasodilation was observed that reversed to a constriction at higher concentrations. Previously we had demonstrated that bovine adrenal cortical arterioles also constrict to the thromboxane analog U46619, serotonin, and endothelin-1 (16). The AII-induced constriction was mediated by the AT1 receptor subtype. This was indicated by blockade of the AII-induced constriction with the AT1 receptor antagonist losartan and verification of AT1 receptor expression in the cortical arteries. It is well established that AII-dependent constrictions in arteries from numerous vascular beds are mediated by the AT1 receptor subtype (14, 25). The presence of losartan enhanced the dilations to AII. Conversely, endothelial cell removal enhanced the constrictions to AII. This suggests that AII-induced constrictions of adrenal cortical arteries are modulated by relaxing factors produced by the endothelium.
Vasodilation by AII was blocked by endothelial cell removal and the NO synthase inhibitor, L-NA, and partially inhibited by the AT2 receptor antagonist, PD123319. Further inhibition of the AII-dependent relaxations with the nonspecific AII antagonist, saralasin, was not observed. These data suggest that AII vasodilation of the cortical arteries is mediated to a large extent by activation of endothelial cell AT2 receptors, resulting in NO production. Using the DAF-2 assay, we demonstrated that AII stimulated NO-dependent fluorescence in the endothelium of the cortical arteries. The AII-induced increase in fluorescence was also blocked by PD123319. AT2 receptor and eNOS protein expression were verified in adrenal endothelial cells and adrenal cortical arteries by Western immunoblot. AT2-dependent vasodilations and relaxations to AII have been observed in numerous arteries including renal, cerebral, mesenteric, and coronary arteries (26, 27, 28, 29, 30). Similar to the adrenal cortical arteries, AII relaxations in coronary arteries were mediated by NO (30). In the kidney, NO is believed to mediate AT2 receptor function (31). Therefore, contribution of NO to the AT2-dependent dilations of the perfused cortical arteries is consistent with results in other vascular beds.
In the adrenal cortical arteries, residual dilations remained after inhibition of AT2 receptors with PD123319. The residual dilations could be due to incomplete blockade of AT2 receptors or the activation of other AII receptor subtypes on the adrenal endothelium not sensitive to inhibition by PD123319 or losartan. In this regard, AT4 receptors have been identified in bovine vascular endothelial cells and bovine adrenal glands (32, 33). However, the role of AT4 receptors in mediating endothelium-dependent dilations to AII are not clear. Additionally, AII vascular activity has been linked to transactivation of receptor tyrosine kinases (34, 35) and a role of receptor tyrosine kinases in the NO-dependent dilations to AII should be considered.
A biphasic response to AII is not unique to the adrenal circulation. In perfused porcine coronary arteries, AT1-dependent constriction to AII was observed at low concentrations and reversed to a AT2-dependent dilation with higher concentrations (29). In the mesenteric vascular bed of the rat, infusion of AII induced a vasodilation followed by a constriction (36). Additionally, AT2-dependent relaxations are often revealed only after selective antagonism of the AT1 receptor (27, 30). Thus, in several vascular beds, AII regulation of vascular tone is complex and involves both AT1 and AT2 receptor subtype activation.
In addition to direct vasodilation and the modulation of AII constriction, the AII-induced NO release could inhibit the secretion of aldosterone. NO binds to cytochrome p450 in adrenal cells and inhibits steroidogenesis (22). Endothelial cell NO partially inhibits AII-stimulation of aldosterone release from cultured bovine ZG cells (22). Because ZG cells are in close proximity to the capsular and subcapsular arterioles, it is possible that AII-stimulated endothelial cell NO acts as a negative paracrine regulator of aldosterone secretion.
Previous in vivo studies showed that AII infusion had either no effect or decreased adrenal blood flow. In sheep with cervical adrenal autotransplants or dexamethasone-treated rabbits, AII infusion did not alter adrenal blood (37, 38, 39, 40, 41). In rats infusions of high concentrations of AII decreased adrenal blood flow (42, 43), which was further reduced by the NO synthase inhibitor, N-omega-nitro-L-arginine methyl ester (43). Thus, it appears AII-induced alterations of adrenal blood flow in vivo are modulated by NO. These findings are in agreement with our findings in the isolated cortical arteries. However, in the in vivo model, the vasodilatory effect of AII and the resulting increase in adrenal blood flow are masked by other physiological effects of the peptide. Because adrenal cortical arteries are a site of myogenic regulation (7), the increase in systemic blood pressure by AII would result in pressure-induced constriction of the adrenal cortical arteries. This constriction could supersede the direct vasodilatory effects of AII.
In the human, normal plasma concentrations of AII are 9–36 pg/ml (8–30 pM) (44, 45, 46). Correspondingly, AII plasma concentrations in unrestrained bovine steers average 55–65 pM (47, 48). These concentrations correlate to a vasodilator effect in adrenal cortical arteries of this study. Increasing circulating AII concentrations 4- to 15-fold above basal values would further dilate the cortical arteries and increase adrenal blood flow. A concentration of 120 pg/ml (10 nM) corresponds to the maximal AII dilatory state of bovine adrenal cortical arteries. Further increases in AII concentrations would stimulate a constrictor response and result in decreased adrenal blood flow. In a small number of active, upright, furosemide-treated hypertensive patients, without evidence of primary aldosteronism, AII plasma concentrations in excess of 200 pg/ml have been measured (44). Thus, high pathophysiological levels of AII would stimulate constriction of adrenal cortical arteries that would limit AII delivery to the adrenal gland and secretion of aldosterone to the circulation.
Acknowledgments
We thank Gretchen Barg for secretarial assistance.
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