Effect of Angiotensin II and ACTH on Adrenal Blood Flow in the Male Rat Adrenal Gland In Vivo

Effect of Angiotensin II and ACTH on Adrenal Blood Flow in the Male Rat Adrenal Gland In Vivo Abstract Angiotensin II (Ang II) and adrenocorticotropic hormone (ACTH) regulate adrenal vascular tone in vitro through endothelial and zona glomerulosa cell–derived mediators. The role of these mediators in regulating adrenal blood flow (ABF) and mean arterial pressure (MAP) was examined in anesthetized rats. Ang II (0.01 to 100 ng/kg) increased ABF [maximal increase of 97.2 ± 6.9 perfusion units (PUs) at 100 ng/kg] and MAP (basal, 115 ± 7 mm Hg; Ang II, 163 ± 5 mm Hg). ACTH (0.1 to 1000 ng/kg) also increased ABF (maximum increase of 91.4 ± 10.7 PU) without changing MAP. ABF increase by Ang II was partially inhibited by the nitric oxide (NO) synthase inhibitor N-nitro-l-arginine methyl ester (L-NAME) (maximum increase of 72.9 ± 4.2 PU), the cytochrome P450 inhibitor miconazole (maximum increase of 39.1 ± 6.8 PU) and the epoxyeicosatrienoic acid (EET) antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) (maximum increase of 56.0 ± 13.7 PU) alone, whereas combined administration of miconazole and L-NAME (maximum increase of 16.40 ± 8.98 PU) ablated it. These treatments had no effect on MAP. Indomethacin did not affect the increase in ABF or MAP induced by Ang II. The ABF increase by ACTH was partially ablated by miconazole and 14,15-EEZE but not by L-NAME. Steroidogenic stimuli such as Ang II and ACTH increase ABF to promote oxygen and cholesterol delivery for steroidogenesis and aldosterone transport to its target tissues. The increases in ABF induced by Ang II are mediated by release of NO and EETs, whereas ABF increases with ACTH are mediated by EETs only. The mammalian adrenal gland has two main structural divisions: the outer cortex and the inner medulla (1). Adrenal cortex consists of the outer cortical zona glomerulosa (ZG) and inner cortical zona fasciculata and the zona reticularis, which synthesize the mineralocorticoid aldosterone and glucocorticoid cortisol, respectively (2, 3). The adrenal gland is extensively vascularized and receives a high proportion of the cardiac output corresponding to its mass (1). The rat adrenal gland, which constitutes ~0.02% of the total body weight, receives 0.14% of the total cardiac output (4, 5). Many arteries emerge from the aorta and the renal and phrenic arteries to supply the adrenal glands. These adrenal arteries penetrate the adrenal capsule to form an anastomotic subcapsular network of resistance arteries within the ZG region (6). The primary site of regulation of adrenal blood flow (ABF) is this capsular and subcapsular arteriolar plexus (1, 2, 7). The vascular network provides a bidirectional communication between the vascular endothelial and smooth muscle cells and adrenocortical ZG cells. For example, endothelial cell–derived paracrine factors affect steroidogenic cells, indicating the existence of complicated intra-adrenal mechanisms that control steroidogenesis and ABF (8–11). For instance, nitric oxide (NO) of vascular endothelial origin inhibits steroidogenesis (10, 12), and an endothelium-derived steroidogenic peptide stimulates aldosterone synthesis (9, 13). Blood flow through the adrenal glands is highly controlled by a number of different neural and hormonal mechanisms (7, 14), such as release of neuropeptides (7) including vasodilators vasoactive intestinal peptide, met-enkephalin, and calcitonin gene–related peptide (15, 16). Steroidogenic stimuli are known to increase ABF, which seems to facilitate steroidogenesis (1, 17). Numerous hormonal and paracrine factors are reported to exert a stimulatory effect on aldosterone secretion, including adrenocorticotropic hormone (ACTH), angiotensin II (Ang II), and potassium. ACTH exerts a stimulatory effect on aldosterone and cortisol secretion and increases ABF in vivo (18–21). In contrast, ACTH does not affect vascular tone of isolated bovine adrenal arterioles in vitro; however, in the presence of ZG cells, ACTH induces relaxation (11). This effect indicates that ZG cells are essential for the ACTH-induced relaxation. The ACTH-induced relaxation is inhibited by cytochrome P450 (CYP) inhibitors, the potassium channel blocker iberiotoxin, and the epoxyeicosatrienoic acid (EET) antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) (11). ACTH stimulates EET release from ZG cells. EETs are CYP metabolites of arachidonic acid (22), and their actions including vasorelaxation are blocked by 14,15-EEZE (11, 23). EETs activate iberiotoxin-sensitive potassium channels of arterial smooth muscle cell, which cause membrane hyperpolarization and subsequent vasorelaxation (24, 25). ACTH induces increase in ABF in anesthetized rats in vivo (26). On the other hand, Ang II causes vasodilation through a direct vascular mechanism and indirect ZG-mediated mechanism. Ang II induces vascular relaxation in vitro in isolated adrenal arteries that is mediated by activation of endothelial angiotensin type 2 receptors and release of NO (25, 27). In addition, Ang II stimulates ZG cell–mediated EET release that also results in vasodilation (25, 27, 28). Like ACTH, Ang II also increases ABF in rats (26). The involvement of these intra-adrenal mechanisms in controlling ABF in vivo is in need of study. Previously, many approaches were adopted to measure ABF including the hydrogen washout technique (29), rubidium fractionation (30), venous outflow (31, 32), radiographic imaging (33), and radiolabeled, fluorescent-labeled, or colored microsphere distribution (7). The major limitation of these methods was their inability to measure ABF continuously. Thus, we developed and described a laser Doppler flowmetry method for fast, real-time, reproducible measurement of continuous changes in ABF in intact animals (26). With this method, Ang II and ACTH produced rapid increases in ABF over a range of concentrations in anesthetized rats. Because in vitro studies indicate that endothelial NO and ZG cell EETs both mediate the dilation to Ang II and ZG cell EETs release to ACTH, we applied the adrenal laser Doppler flowmetry method to test the role of these mediators of Ang II and ACTH on ABF in vivo using inhibitors of prostaglandins (PGs), NO, and EET pathways. Materials and Methods Materials ACTH, Ang II, N-nitro-l-arginine methyl ester (L-NAME), indomethacin, and miconazole were purchased from Sigma-Aldrich. 14,15-EEZE was synthesized by Dr. Falck. Miconazole was dissolved in dimethylsulfoxide, followed by dilution in sterile saline. Indomethacin was dissolved in 0.05 M sodium carbonate. All other drugs used were dissolved in sterile saline. Animal preparation As described earlier (26), experiments were performed on male Sprague Dawley rats (250 to 300 g). Animals were allowed food and water ad libitum before experimentation. The rats were anesthetized with pentobarbital (50 to 60 mg/kg intraperitoneally followed by 30 mg/kg/h intravenously as needed). The left femoral artery and vein were cannulated and used for mean arterial pressure (MAP) measurements and drug administration. We exposed the left adrenal gland by making a small abdominal incision just under the left thoracic cage. Protocols for the experiments were approved by the Animal Care Committee of the Medical College of Wisconsin, and procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Measurement of ABF and MAP in anesthetized rats Laser Doppler flowmetry (Periflux system 5000; Perimed) was used for measurement of ABF (26). A stainless steel probe (PF-403, 1 mm diameter, 80 mm length, measuring depth 1 mm) was placed vertically above the exposed adrenal gland. A micromanipulator was used for appropriate and proper positioning. Laser Doppler shift indicated measurement of ABF, which was expressed in perfusion units (PUs) (34, 35). MAP was recorded from the femoral artery through a pressure transducer coupled with Powerlab 4/25 data acquisition system (ADInstruments) and analyzed with Chart software (ADInstruments). Before we tested the effect of Ang II and ACTH, rats were stabilized for 1 hour. Afterward, bolus intravenous injections of Ang II (0.01 to 100 ng/kg) and ACTH (0.1 to 1000 ng/kg) were made, followed by a flush of the cannula with 0.1 mL saline. The effect of each dose of Ang II and ACTH on ABF was continuously recorded for 5 to 10 minutes. To see the effect of inhibitors of PGs, NO, and EETs on ABF, an intravenous injection of indomethacin (5 mg/kg intraperitoneally), L-NAME (10 mg/kg), miconazole (2 mg/kg), 14,15-EEZE (2.5 mg/kg), or vehicle was given. Sequential intravenous injection of Ang II and ACTH was given after 10 minutes of administration of inhibitors. The effect of each dose of Ang II and ACTH on ABF was continuously recorded for 5 to 10 minutes. Measurements of plasma eicosanoids in anesthetized rats treated with inhibitors A separate series of anesthetized rats were surgically prepared as described above. After equilibration for 1 hour, they were treated with vehicle, miconazole, or indomethacin. After 30 minutes, blood was removed from the vena cava, placed on ice, and centrifuged at 4°C, and the plasma collected. The plasma was extracted with C18 extraction columns and analyzed for cyclooxygenase [prostaglandin E2 (PGE2) and thromboxane B2 (TxB2)], lipoxygenase [15-hydroxyeicosatetraenoic acid (15-HETE)], and CYP epoxygenase [14,15-EET and 11,12-EET and dihydroxyeicosatrienoic acids (DHETs)] metabolites of arachidonic acid by liquid chromatography mass spectrometry as previously described (36, 37). Statistical analysis Data are presented as mean ± standard error of the mean. Experiments and treatments were repeated in groups of 5 to 11 rats. Significance between two groups was evaluated by Student t test. Significance between and within multiple groups was evaluated by analysis of variance followed by the Dunnett Multiple Comparison Test. P > 0.05 was considered significant. Results Effect of Ang II on ABF Intravenous injection of Ang II (0.01 to 100 ng/kg) increased ABF (maximal increase of 97.2 ± 9.2 PU with 100 ng/kg) (Fig. 1A). All dosages increased ABF. Ang II increased MAP in a dose-dependent manner, with maximal MAP of 163.3 ± 5.6 mm Hg with 100 ng Ang II/kg (Fig. 1B). The time course of ABF responses to Ang II (1 ng/kg) showed a gradual increase that plateaued at 1 to 2 minutes (Fig. 1C). ABF returned to basal levels in 4 to 5 minutes. The NO synthase inhibitor L-NAME attenuated, but did not block, the increase in ABF to Ang II from 97.2 ± 9.2 PU to 72.9 ± 4.2 PU. The Ang II increase in MAP was not affected by pretreatment with L-NAME (Fig. 1A). Thus, NO contributes to Ang II–induced increases in ABF. Figure 1. View largeDownload slide Effect of Ang II on (A) ABF and (B) MAP in the absence (control) and presence of L-NAME (10 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is significantly different from basal. (C) Time course of ABF changes in response to a bolus injection of Ang II (1 ng/kg). The 10-second mean values for change in ABF are indicated for the first 2 minutes, followed by the 60-second mean values up to 4 minutes. Each value represents the mean ± standard error of the mean for n = 6 to 9 rats. *P < 0.05 compared with control. Figure 1. View largeDownload slide Effect of Ang II on (A) ABF and (B) MAP in the absence (control) and presence of L-NAME (10 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is significantly different from basal. (C) Time course of ABF changes in response to a bolus injection of Ang II (1 ng/kg). The 10-second mean values for change in ABF are indicated for the first 2 minutes, followed by the 60-second mean values up to 4 minutes. Each value represents the mean ± standard error of the mean for n = 6 to 9 rats. *P < 0.05 compared with control. The Ang II increase in ABF and MAP was not affected by indomethacin (5 mg/kg) pretreatment (Fig. 2A and 2B). However, the Ang II–induced increase in ABF was significantly (P < 0.05) decreased with the CYP inhibitor miconazole (2 mg/kg), and the combination of miconazole and L-NAME pretreatment further attenuated the increase in ABF from 96.4 ± 10.0 PU to 39.1 ± 6.8 PU and 16.4 ± 8.9 PU, respectively (Fig. 3A). Ang II increased MAP in a dose-dependent manner, with maximal MAP of 162.7 ± 8.3 mm Hg with 100 ng Ang II/kg (Fig. 3C). The Ang II increase in MAP was not affected by miconazole (2 mg/kg) or the combined administration of miconazole and L-NAME (10 mg/kg). The EET antagonist 14,15-EEZE (2.5 mg/kg) also attenuated the Ang II increase in ABF from 96.6 ± 11.0 PU to 45.0 ± 10.5 PU (Fig. 3B). 14,15-EEZE did not affect MAP (Fig. 3D). Figure 2. View largeDownload slide Effect of Ang II on (A) ABF and (B) MAP in the absence (control) and presence of indomethacin (5 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is not significantly different from basal. Each value represents the mean ± standard error of the mean for n = 7 rats. Figure 2. View largeDownload slide Effect of Ang II on (A) ABF and (B) MAP in the absence (control) and presence of indomethacin (5 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is not significantly different from basal. Each value represents the mean ± standard error of the mean for n = 7 rats. Figure 3. View largeDownload slide Effects of Ang II on (A and B) ABF and (C and D) MAP (A and C) in the absence (control) or presence of miconazole (2.5 mg/kg) and of miconazole and L-NAME (10 mg/kg) and (B and D) in the absence or presence of 14,15-EEZE (2.5 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is significantly different from basal. Each value represents the mean ± standard error of the mean for n = 7 to 11 rats. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control. Figure 3. View largeDownload slide Effects of Ang II on (A and B) ABF and (C and D) MAP (A and C) in the absence (control) or presence of miconazole (2.5 mg/kg) and of miconazole and L-NAME (10 mg/kg) and (B and D) in the absence or presence of 14,15-EEZE (2.5 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is significantly different from basal. Each value represents the mean ± standard error of the mean for n = 7 to 11 rats. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control. Effect of ACTH on ABF We also studied the effect of ACTH on ABF. Intravenous injection of ACTH increased ABF at all dosages tested (maximal increase = 91.4 ± 10.8 PU with 1000 ng ACTH/kg) (Fig. 4A). ACTH did not alter MAP (Fig. 4B). The time course of ABF responses to ACTH (1 ng/kg) shows that ABF increased and plateaued in 1.0 to 1.7 minutes and returned to basal levels within 4 to 5 minutes (Fig. 4C). The injection of saline (0.1 mL) alone did not alter ABF. The ACTH-induced increase in ABF was not affected by L-NAME (10 mg/kg) pretreatment (Fig. 4A) or by indomethacin (data not shown). The increase in ABF was decreased by pretreatment with the CYP inhibitor miconazole (2 mg/kg) from 91.4 ± 4.7 PU to 66.0 ± 8.9 PU (Fig. 5A). However, this decrease in ABF with miconazole was not further changed with the combination of miconazole and L-NAME (Fig. 5A). Miconazole alone did not alter ABF. Pretreatment of the rat with 14,15-EEZE (2.5 mg/kg) did not affect the ACTH increase in ABF at lower dosages (0.1 to 1 ng/kg) but eliminated the increase in ABF at higher dosages (10 to 1000 ng/kg) (Fig. 5B). Thus, PGs do not appear to contribute to the increase in ABF to Ang II and ACTH. However, the studies with L-NAME show that NO contributes to the ABF increase with Ang II but not with ACTH. CYP metabolites of arachidonic acid, the EETs, contribute to the increase in ABF with Ang II and ACTH. Both EETs and NO in combination mediate the ABF increase with Ang II. Figure 4. View largeDownload slide Effects of ACTH on (A) ABF and (B) MAP in the absence (control) and presence of L-NAME (10 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is significantly different from basal. (C) Time course of ABF changes in response to a bolus injection of ACTH (1 ng/kg). The 10-second mean values for change in ABF are indicated for the first 2 minutes, followed by the 60-second mean values up to 4 minutes. Each value represents the mean ± standard error of the mean for n = 5 to 10 rats. Figure 4. View largeDownload slide Effects of ACTH on (A) ABF and (B) MAP in the absence (control) and presence of L-NAME (10 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is significantly different from basal. (C) Time course of ABF changes in response to a bolus injection of ACTH (1 ng/kg). The 10-second mean values for change in ABF are indicated for the first 2 minutes, followed by the 60-second mean values up to 4 minutes. Each value represents the mean ± standard error of the mean for n = 5 to 10 rats. Figure 5. View largeDownload slide Effects of ACTH on (A and B) ABF and (C and D) MAP (A and C) in the absence (control) or presence of miconazole (2.5 mg/kg) and of miconazole and L-NAME (10 mg/kg) and (B and D) in the absence or presence of 14,15-EEZE (2.5 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is significantly different from basal. Each value represents the mean ± standard error of the mean for n = 7 to 11 rats. *P < 0.05, **P < 0.01 compared with control. Figure 5. View largeDownload slide Effects of ACTH on (A and B) ABF and (C and D) MAP (A and C) in the absence (control) or presence of miconazole (2.5 mg/kg) and of miconazole and L-NAME (10 mg/kg) and (B and D) in the absence or presence of 14,15-EEZE (2.5 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is significantly different from basal. Each value represents the mean ± standard error of the mean for n = 7 to 11 rats. *P < 0.05, **P < 0.01 compared with control. Effect of inhibitors on plasma eicosanoids Plasma PGE2 and TxB2, 15-HETE and 14,15-EET and 11,12-EET, and DHET were used as indices of the activity of the cyclooxygenase, lipoxygenase and CYP epoxygenase pathways of arachidonic acid metabolism, respectively (Table 1). Compared with control, vehicle-treated rats, the plasma concentrations of TxB2 and PGE2 were decreased significantly with indomethacin. The plasma concentrations of 15-HETE, EETs, and DHETs were similar between vehicle- and indomethacin-treated rats. Miconazole significantly decreased the plasma concentrations of EETs and DHETs without changing 15-HETE. The plasma concentrations of TxB2 and PGE2 were decreased with miconazole; however, these decreases did not attain statistical significance. These data support the inhibition of cyclooxygenase by indomethacin and CYP epoxygenase by miconazole in the in vivo dosages used. Furthermore, the failure of indomethacin to alter the ABF changes with Ang II and ACTH occurred in the presence of documented cyclooxygenase inhibition by the drug. Thus, cyclooxygenase metabolites do not contribute to Ang II- and ACTH- mediated changes in ABF. These data also indicate that miconazole inhibits the ABF increases to Ang II and ACTH by suppressing CYP epoxygenase activity and EET and DHET synthesis. Though acting at different sites in the CYP-EET pathway, 14,15-EEZE and miconazole had similar effects. Table 1. Effect of Indomethacin and Miconazole Treatment on Plasma Concentrations of Selected Eicosanoids   Plasma Concentration (ng/mL)  Eicosanoid  Control  Indomethacin  Miconazole  TxB2  2.57 ± 1.52  0.00 ± 0.00a  1.52 ± 1.02  PGE2  0.05 ± 0.01  0.0015 ± 0.001b  0.024 ± 0.013  15-HETE  2.82 ± 0.60  2.77 ± 1.03  2.38 ± 0.90  11,12-DHET  0.55 ± 0.05  0.62 ± 0.03  0.07 ± 0.04c  14,15-DHET  0.52 ± 0.05  0.54 ± 0.05  0.21 ± 0.03c  11,12-EET  0.42 ± 0.06  0.48 ± 0.06  0.22 ± 0.03a  14,15-EET  0.84 ± 0.10  0.85 ± 0.23  0.41 ± 0.08a    Plasma Concentration (ng/mL)  Eicosanoid  Control  Indomethacin  Miconazole  TxB2  2.57 ± 1.52  0.00 ± 0.00a  1.52 ± 1.02  PGE2  0.05 ± 0.01  0.0015 ± 0.001b  0.024 ± 0.013  15-HETE  2.82 ± 0.60  2.77 ± 1.03  2.38 ± 0.90  11,12-DHET  0.55 ± 0.05  0.62 ± 0.03  0.07 ± 0.04c  14,15-DHET  0.52 ± 0.05  0.54 ± 0.05  0.21 ± 0.03c  11,12-EET  0.42 ± 0.06  0.48 ± 0.06  0.22 ± 0.03a  14,15-EET  0.84 ± 0.10  0.85 ± 0.23  0.41 ± 0.08a  Data are presented as means ± standard error of the mean of four experiments. Two-tailed Student t test was used for determining the significance of observed differences between experimental values, with P < 0.05 considered statistically significant. a P < 0.05. b P < 0.01. c P < 0.005. View Large Discussion ABF is critical to the supply of oxygen, nutrients, and cholesterol for steroidogenesis and delivery of aldosterone and cortisol to their target organs (1, 2, 7). The regulation of ABF by steroidogenic stimuli and the intra-adrenal mediators of adrenal vascular tone are poorly understood. We developed an ABF method using laser Doppler flowmetry in anesthetized rats and demonstrated that Ang II and ACTH increase ABF (26). Using this method, this investigation characterized the effects of Ang II and ACTH on ABF in vivo and extended the previous investigations by describing the endogenous mediators involved. Earlier studies described conflicting effects of Ang II on ABF that may be attributed to differences in methods, Ang II dosage, or species studied. For instance, Ang II increased ABF in anephric rats (38). However, Ang II in higher concentrations reduced ABF (38, 39). These decreases in ABF to Ang II were increased by pretreatment with L-NAME, indicating that endogenous NO limits Ang II constriction (39). Also, in dexamethasone-treated rabbits and sheep with cervical adrenal autotransplant, Ang II had no effect on ABF (40, 41). The effect of ACTH on ABF has also been investigated in experimental animals (1). Similarly, controversy exists concerning the dosage and timing of the ABF increase with ACTH. For instance, physiological concentrations of ACTH did not affect ABF, but concentrations above the physiological range increased ABF (18, 19). ACTH produced an immediate increase in flow in the intact perfused canine adrenal gland in one study (20), whereas in another study, a delayed dilation to ACTH was observed (42, 43). ACTH also increased ABF in perfused fetal sheep adrenal glands (21). Intra-adrenal mediators of the ABF responses to Ang II and ACTH were not investigated in detail previously. However, in in vitro studies, dilation to Ang II was mediated by NO and EETs, whereas dilation to ACTH was mediated by EETs only (25, 27, 44) (Fig. 6). We tested these possibilities in vivo by using inhibitors of PG, NO, and EET synthesis. Intravenous injection of both Ang II and ACTH dose-dependently increased ABF. Ang II also increases MAP, but ACTH did not. The cyclooxygenase inhibitor indomethacin, in a dosage that significantly decreased plasma concentration of PGE2 and TxB2, had no effect on the Ang II increase in ABF, so PGs do not contribute to the increase in ABF to Ang II. This finding is consistent with the findings of in vitro studies in isolated adrenal arteries and ZG cells that indomethacin did not affect ZG cell–mediated vasorelaxations to Ang II (25). Increase in ABF to Ang II was inhibited 60% with L-NAME pretreatment, but L-NAME did not affect the ABF increase to ACTH. L-NAME did not affect the increase in MAP to Ang II, so changes in MAP could not explain the inhibition of ABF. These findings are consistent with the findings of previous in vitro studies in adrenal arteries that endothelial NO regulates relaxation to Ang II but not ACTH (25, 27) (Fig. 6). In isolated adrenal arteries, ACTH induces relaxation only in the presence of ZG cells. These relaxations to ACTH were not affected by endothelium removal or NO synthase inhibition. Figure 6. View largeDownload slide Summary of the regulation of adrenal vascular tone and ABF by Ang II and ACTH. Ang II–mediated vasodilation and increase in ABF are mediated by endothelial NO and ZG cell EETs and DHETs, whereas ACTH is mediated by ZG cell EETs and DHETs only. Arg, arginine; AT2, Ang II type 2 receptor; AT?, Ang II receptor, not type 1 or 2; cGMP, cyclic guanosine monophosphate; EETr, EET receptor; Gs, guanine nucleotide binding protein αs; GTP, guanosine triphosphate; K+, potassium; MCR, melanocortin receptor; NOS, NO synthase; PL, phospholipid; sGC, soluble guanylyl cyclase. Figure 6. View largeDownload slide Summary of the regulation of adrenal vascular tone and ABF by Ang II and ACTH. Ang II–mediated vasodilation and increase in ABF are mediated by endothelial NO and ZG cell EETs and DHETs, whereas ACTH is mediated by ZG cell EETs and DHETs only. Arg, arginine; AT2, Ang II type 2 receptor; AT?, Ang II receptor, not type 1 or 2; cGMP, cyclic guanosine monophosphate; EETr, EET receptor; Gs, guanine nucleotide binding protein αs; GTP, guanosine triphosphate; K+, potassium; MCR, melanocortin receptor; NOS, NO synthase; PL, phospholipid; sGC, soluble guanylyl cyclase. CYP inhibitor miconazole and the EET antagonist 14,15-EEZE inhibit ABF increases to Ang II and ACTH in vivo. The inhibitors did not affect MAP, indicating that changes in MAP cannot explain the reduced ABF increases to Ang II or ACTH. Previous studies in isolated adrenal arteries showed that Ang II stimulates endothelial cell Ang type 2 receptors that mediate release of NO (27, 28) (Fig. 6). Additionally, Ang II activates ZG cells that release EET to induce relaxation (25). ACTH does not affect vascular tone in isolated adrenal arterioles (44) but induces relaxation in presence of ZG cells (11). CYP inhibitors (miconazole and SKF525a), high K+, iberiotoxin, and 14,15-EEZE attenuated the relaxations to ACTH in vitro (11). In a previous study, miconazole blocked the ABF increase to a single dose of Ang II and ACTH (26). In the current study, miconazole attenuated but did not block the ABF increases to a range of concentrations of Ang II and ACTH. The reasons for these differences in ABF inhibition by miconazole are not known. However, these studies with miconazole support a role for CYP metabolites of arachidonic acid in the regulation of ABF. This conclusion is supported by miconazole causing a significant decrease in the plasma concentrations of 14,15-EET, 11,12-EET, and DHET. Additionally, the decrease in ABF to Ang II and ACTH with 14,15-EEZE indicates that the CYP metabolites are arachidonic acid–derived EETs and that ZG cell–derived EETs mediate a portion of the relaxation to Ang II and relaxations to ACTH in vivo. The combination of L-NAME and miconazole completely eliminated the increase in ABF to Ang II; however, the effect of this combination on the increase in ABF to ACTH was similar to that of miconazole alone. These findings indicate that combined mediators, endothelial NO and ZG cell–derived EETs, are involved in the increase in ABF to Ang II; however, only ZG cell–derived EETs explain the increase in ABF to ACTH in both in vivo and in vitro (Fig. 6). In studies in isolated coronary arteries, the endothelium-dependent relaxations to bradykinin were mediated by NO and EETs; however, the CYP and EET component was not apparent until NO synthase was inhibited (45–47). In these arteries, NO reduced CYP activity, so inhibition of NO synthesis restored EET synthesis. Such an interaction was not observed in adrenal arteries or adrenal arteries and ZG cells with Ang II (25, 27, 28). Also, ABF increases with Ang II have both NO and EET components that act in parallel. This effect may be due to NO and EETs being synthesized by different cells in the adrenal gland, EETs by ZG cells and NO by endothelial cells (Fig. 6). Unlike endothelial cells, ZG cells do not contain NO synthase or synthesize NO, but both cell types synthesize EETs (11, 48, 49). Apparently, endothelial NO does not inhibit ZG cell EET synthesis, allowing the two mediators to act in parallel. Additionally, Ang II and bradykinin differ in their cellular actions and the endothelial mediators that are released. Bradykinin stimulates both NO and EET production from the coronary vascular endothelium (45, 46), whereas Ang II stimulates only NO production via endothelial Ang II receptor of adrenal arteries (25, 27, 28). The cellular pathways mediating these differences have not been described. Ang II causes vasoconstriction, whereas ACTH has no effects on vascular tone (11, 50). To maintain ABF in the presence of Ang II, endothelial NO is important to counteract the vasoconstriction and maintain or increase ABF (39). Ang II also acts on ZG cells to release EETs, promoting vasodilation and increases in ABF associated with stimulation of steroidogenesis. Because ACTH has no direct vascular effects (11), NO is not needed to oppose constriction, and only ZG cell–derived EETs are needed to link increases in ABF to steroidogenesis. In addition to EET release, Ang II and ACTH stimulate ZG cells to release aldosterone and corticosterone. Miconazole inhibits the synthesis of steroids and EETs (51, 52). However, steroids are not mediators of Ang II- or ACTH-induced dilation in vitro or ABF increases in vivo. Aldosterone and the glucocorticoid dexamethasone did not change vascular tone in vitro in isolated adrenal arteries (11). Also, 14,15-EEZE, like miconazole, attenuated the relaxations to Ang II and ACTH in vitro in isolated adrenal arteries as well as ABF increases in vivo, but unlike miconazole, 14,15-EEZE did not affect steroidogenesis (25). These studies indicate the importance of EETs and NO in the regulation of ABF by Ang II and EETs with ACTH in vivo (Fig. 6). From in vivo studies, it is not know with certainty whether the EETs are derived from ZG cells. However, in vitro studies indicate that ZG cells synthesize and release EETs in response to these stimuli. These studies indicate the importance of NO and EETs in increasing ABF with Ang II and EETs with ACTH and provide in vivo evidence that CYP metabolites of arachidonic acid, the EETs, mediate increases in ABF that are coupled to steroidogenesis. This ZG cell–EET pathway couples an increase in ABF with stimulation of steroidogenesis and promotes the increased delivery of oxygen, cholesterol, and nutrients for steroid synthesis and delivery of newly synthesized steroids to their target tissues. Abbreviations: 14,15-EEZE 14,15-epoxyeicosa-5(Z)-enoic acid 15-HETE 15-hydroxyeicosatetraenoic acid ABF adrenal blood flow ACTH adrenocorticotropic hormone Ang II angiotensin II CYP cytochrome P450 DHET dihydroxyeicosatrienoic acid EET epoxyeicosatrienoic acid L-NAME N-nitro-l-arginine methyl ester MAP mean arterial pressure NO nitric oxide PG prostaglandin PGE2 prostaglandin E2 PU perfusion unit TxB2 thromboxane B2 ZG zona glomerulosa. Acknowledgments The authors thank Gretchen Barg for her secretarial assistance. This research was conducted while Dr. Abdul Jabbar Shah was on sabbatical leave from COMSATS Institute of Information Technology, Abbottabad, Pakistan. Financial Support: These studies were supported by National Heart, Lung, and Blood Institute Grant HL-83297 (to W.B.C.); a Ralph and Marian Falk Medical Research Trust Bank of America, N.A., Trustee Grant (to J.R.F.); and Robert A. Welch Foundation Grant I-0011 (to J.R.F.). 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Effect of Angiotensin II and ACTH on Adrenal Blood Flow in the Male Rat Adrenal Gland In Vivo

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Abstract

Abstract Angiotensin II (Ang II) and adrenocorticotropic hormone (ACTH) regulate adrenal vascular tone in vitro through endothelial and zona glomerulosa cell–derived mediators. The role of these mediators in regulating adrenal blood flow (ABF) and mean arterial pressure (MAP) was examined in anesthetized rats. Ang II (0.01 to 100 ng/kg) increased ABF [maximal increase of 97.2 ± 6.9 perfusion units (PUs) at 100 ng/kg] and MAP (basal, 115 ± 7 mm Hg; Ang II, 163 ± 5 mm Hg). ACTH (0.1 to 1000 ng/kg) also increased ABF (maximum increase of 91.4 ± 10.7 PU) without changing MAP. ABF increase by Ang II was partially inhibited by the nitric oxide (NO) synthase inhibitor N-nitro-l-arginine methyl ester (L-NAME) (maximum increase of 72.9 ± 4.2 PU), the cytochrome P450 inhibitor miconazole (maximum increase of 39.1 ± 6.8 PU) and the epoxyeicosatrienoic acid (EET) antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) (maximum increase of 56.0 ± 13.7 PU) alone, whereas combined administration of miconazole and L-NAME (maximum increase of 16.40 ± 8.98 PU) ablated it. These treatments had no effect on MAP. Indomethacin did not affect the increase in ABF or MAP induced by Ang II. The ABF increase by ACTH was partially ablated by miconazole and 14,15-EEZE but not by L-NAME. Steroidogenic stimuli such as Ang II and ACTH increase ABF to promote oxygen and cholesterol delivery for steroidogenesis and aldosterone transport to its target tissues. The increases in ABF induced by Ang II are mediated by release of NO and EETs, whereas ABF increases with ACTH are mediated by EETs only. The mammalian adrenal gland has two main structural divisions: the outer cortex and the inner medulla (1). Adrenal cortex consists of the outer cortical zona glomerulosa (ZG) and inner cortical zona fasciculata and the zona reticularis, which synthesize the mineralocorticoid aldosterone and glucocorticoid cortisol, respectively (2, 3). The adrenal gland is extensively vascularized and receives a high proportion of the cardiac output corresponding to its mass (1). The rat adrenal gland, which constitutes ~0.02% of the total body weight, receives 0.14% of the total cardiac output (4, 5). Many arteries emerge from the aorta and the renal and phrenic arteries to supply the adrenal glands. These adrenal arteries penetrate the adrenal capsule to form an anastomotic subcapsular network of resistance arteries within the ZG region (6). The primary site of regulation of adrenal blood flow (ABF) is this capsular and subcapsular arteriolar plexus (1, 2, 7). The vascular network provides a bidirectional communication between the vascular endothelial and smooth muscle cells and adrenocortical ZG cells. For example, endothelial cell–derived paracrine factors affect steroidogenic cells, indicating the existence of complicated intra-adrenal mechanisms that control steroidogenesis and ABF (8–11). For instance, nitric oxide (NO) of vascular endothelial origin inhibits steroidogenesis (10, 12), and an endothelium-derived steroidogenic peptide stimulates aldosterone synthesis (9, 13). Blood flow through the adrenal glands is highly controlled by a number of different neural and hormonal mechanisms (7, 14), such as release of neuropeptides (7) including vasodilators vasoactive intestinal peptide, met-enkephalin, and calcitonin gene–related peptide (15, 16). Steroidogenic stimuli are known to increase ABF, which seems to facilitate steroidogenesis (1, 17). Numerous hormonal and paracrine factors are reported to exert a stimulatory effect on aldosterone secretion, including adrenocorticotropic hormone (ACTH), angiotensin II (Ang II), and potassium. ACTH exerts a stimulatory effect on aldosterone and cortisol secretion and increases ABF in vivo (18–21). In contrast, ACTH does not affect vascular tone of isolated bovine adrenal arterioles in vitro; however, in the presence of ZG cells, ACTH induces relaxation (11). This effect indicates that ZG cells are essential for the ACTH-induced relaxation. The ACTH-induced relaxation is inhibited by cytochrome P450 (CYP) inhibitors, the potassium channel blocker iberiotoxin, and the epoxyeicosatrienoic acid (EET) antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) (11). ACTH stimulates EET release from ZG cells. EETs are CYP metabolites of arachidonic acid (22), and their actions including vasorelaxation are blocked by 14,15-EEZE (11, 23). EETs activate iberiotoxin-sensitive potassium channels of arterial smooth muscle cell, which cause membrane hyperpolarization and subsequent vasorelaxation (24, 25). ACTH induces increase in ABF in anesthetized rats in vivo (26). On the other hand, Ang II causes vasodilation through a direct vascular mechanism and indirect ZG-mediated mechanism. Ang II induces vascular relaxation in vitro in isolated adrenal arteries that is mediated by activation of endothelial angiotensin type 2 receptors and release of NO (25, 27). In addition, Ang II stimulates ZG cell–mediated EET release that also results in vasodilation (25, 27, 28). Like ACTH, Ang II also increases ABF in rats (26). The involvement of these intra-adrenal mechanisms in controlling ABF in vivo is in need of study. Previously, many approaches were adopted to measure ABF including the hydrogen washout technique (29), rubidium fractionation (30), venous outflow (31, 32), radiographic imaging (33), and radiolabeled, fluorescent-labeled, or colored microsphere distribution (7). The major limitation of these methods was their inability to measure ABF continuously. Thus, we developed and described a laser Doppler flowmetry method for fast, real-time, reproducible measurement of continuous changes in ABF in intact animals (26). With this method, Ang II and ACTH produced rapid increases in ABF over a range of concentrations in anesthetized rats. Because in vitro studies indicate that endothelial NO and ZG cell EETs both mediate the dilation to Ang II and ZG cell EETs release to ACTH, we applied the adrenal laser Doppler flowmetry method to test the role of these mediators of Ang II and ACTH on ABF in vivo using inhibitors of prostaglandins (PGs), NO, and EET pathways. Materials and Methods Materials ACTH, Ang II, N-nitro-l-arginine methyl ester (L-NAME), indomethacin, and miconazole were purchased from Sigma-Aldrich. 14,15-EEZE was synthesized by Dr. Falck. Miconazole was dissolved in dimethylsulfoxide, followed by dilution in sterile saline. Indomethacin was dissolved in 0.05 M sodium carbonate. All other drugs used were dissolved in sterile saline. Animal preparation As described earlier (26), experiments were performed on male Sprague Dawley rats (250 to 300 g). Animals were allowed food and water ad libitum before experimentation. The rats were anesthetized with pentobarbital (50 to 60 mg/kg intraperitoneally followed by 30 mg/kg/h intravenously as needed). The left femoral artery and vein were cannulated and used for mean arterial pressure (MAP) measurements and drug administration. We exposed the left adrenal gland by making a small abdominal incision just under the left thoracic cage. Protocols for the experiments were approved by the Animal Care Committee of the Medical College of Wisconsin, and procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Measurement of ABF and MAP in anesthetized rats Laser Doppler flowmetry (Periflux system 5000; Perimed) was used for measurement of ABF (26). A stainless steel probe (PF-403, 1 mm diameter, 80 mm length, measuring depth 1 mm) was placed vertically above the exposed adrenal gland. A micromanipulator was used for appropriate and proper positioning. Laser Doppler shift indicated measurement of ABF, which was expressed in perfusion units (PUs) (34, 35). MAP was recorded from the femoral artery through a pressure transducer coupled with Powerlab 4/25 data acquisition system (ADInstruments) and analyzed with Chart software (ADInstruments). Before we tested the effect of Ang II and ACTH, rats were stabilized for 1 hour. Afterward, bolus intravenous injections of Ang II (0.01 to 100 ng/kg) and ACTH (0.1 to 1000 ng/kg) were made, followed by a flush of the cannula with 0.1 mL saline. The effect of each dose of Ang II and ACTH on ABF was continuously recorded for 5 to 10 minutes. To see the effect of inhibitors of PGs, NO, and EETs on ABF, an intravenous injection of indomethacin (5 mg/kg intraperitoneally), L-NAME (10 mg/kg), miconazole (2 mg/kg), 14,15-EEZE (2.5 mg/kg), or vehicle was given. Sequential intravenous injection of Ang II and ACTH was given after 10 minutes of administration of inhibitors. The effect of each dose of Ang II and ACTH on ABF was continuously recorded for 5 to 10 minutes. Measurements of plasma eicosanoids in anesthetized rats treated with inhibitors A separate series of anesthetized rats were surgically prepared as described above. After equilibration for 1 hour, they were treated with vehicle, miconazole, or indomethacin. After 30 minutes, blood was removed from the vena cava, placed on ice, and centrifuged at 4°C, and the plasma collected. The plasma was extracted with C18 extraction columns and analyzed for cyclooxygenase [prostaglandin E2 (PGE2) and thromboxane B2 (TxB2)], lipoxygenase [15-hydroxyeicosatetraenoic acid (15-HETE)], and CYP epoxygenase [14,15-EET and 11,12-EET and dihydroxyeicosatrienoic acids (DHETs)] metabolites of arachidonic acid by liquid chromatography mass spectrometry as previously described (36, 37). Statistical analysis Data are presented as mean ± standard error of the mean. Experiments and treatments were repeated in groups of 5 to 11 rats. Significance between two groups was evaluated by Student t test. Significance between and within multiple groups was evaluated by analysis of variance followed by the Dunnett Multiple Comparison Test. P > 0.05 was considered significant. Results Effect of Ang II on ABF Intravenous injection of Ang II (0.01 to 100 ng/kg) increased ABF (maximal increase of 97.2 ± 9.2 PU with 100 ng/kg) (Fig. 1A). All dosages increased ABF. Ang II increased MAP in a dose-dependent manner, with maximal MAP of 163.3 ± 5.6 mm Hg with 100 ng Ang II/kg (Fig. 1B). The time course of ABF responses to Ang II (1 ng/kg) showed a gradual increase that plateaued at 1 to 2 minutes (Fig. 1C). ABF returned to basal levels in 4 to 5 minutes. The NO synthase inhibitor L-NAME attenuated, but did not block, the increase in ABF to Ang II from 97.2 ± 9.2 PU to 72.9 ± 4.2 PU. The Ang II increase in MAP was not affected by pretreatment with L-NAME (Fig. 1A). Thus, NO contributes to Ang II–induced increases in ABF. Figure 1. View largeDownload slide Effect of Ang II on (A) ABF and (B) MAP in the absence (control) and presence of L-NAME (10 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is significantly different from basal. (C) Time course of ABF changes in response to a bolus injection of Ang II (1 ng/kg). The 10-second mean values for change in ABF are indicated for the first 2 minutes, followed by the 60-second mean values up to 4 minutes. Each value represents the mean ± standard error of the mean for n = 6 to 9 rats. *P < 0.05 compared with control. Figure 1. View largeDownload slide Effect of Ang II on (A) ABF and (B) MAP in the absence (control) and presence of L-NAME (10 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is significantly different from basal. (C) Time course of ABF changes in response to a bolus injection of Ang II (1 ng/kg). The 10-second mean values for change in ABF are indicated for the first 2 minutes, followed by the 60-second mean values up to 4 minutes. Each value represents the mean ± standard error of the mean for n = 6 to 9 rats. *P < 0.05 compared with control. The Ang II increase in ABF and MAP was not affected by indomethacin (5 mg/kg) pretreatment (Fig. 2A and 2B). However, the Ang II–induced increase in ABF was significantly (P < 0.05) decreased with the CYP inhibitor miconazole (2 mg/kg), and the combination of miconazole and L-NAME pretreatment further attenuated the increase in ABF from 96.4 ± 10.0 PU to 39.1 ± 6.8 PU and 16.4 ± 8.9 PU, respectively (Fig. 3A). Ang II increased MAP in a dose-dependent manner, with maximal MAP of 162.7 ± 8.3 mm Hg with 100 ng Ang II/kg (Fig. 3C). The Ang II increase in MAP was not affected by miconazole (2 mg/kg) or the combined administration of miconazole and L-NAME (10 mg/kg). The EET antagonist 14,15-EEZE (2.5 mg/kg) also attenuated the Ang II increase in ABF from 96.6 ± 11.0 PU to 45.0 ± 10.5 PU (Fig. 3B). 14,15-EEZE did not affect MAP (Fig. 3D). Figure 2. View largeDownload slide Effect of Ang II on (A) ABF and (B) MAP in the absence (control) and presence of indomethacin (5 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is not significantly different from basal. Each value represents the mean ± standard error of the mean for n = 7 rats. Figure 2. View largeDownload slide Effect of Ang II on (A) ABF and (B) MAP in the absence (control) and presence of indomethacin (5 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is not significantly different from basal. Each value represents the mean ± standard error of the mean for n = 7 rats. Figure 3. View largeDownload slide Effects of Ang II on (A and B) ABF and (C and D) MAP (A and C) in the absence (control) or presence of miconazole (2.5 mg/kg) and of miconazole and L-NAME (10 mg/kg) and (B and D) in the absence or presence of 14,15-EEZE (2.5 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is significantly different from basal. Each value represents the mean ± standard error of the mean for n = 7 to 11 rats. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control. Figure 3. View largeDownload slide Effects of Ang II on (A and B) ABF and (C and D) MAP (A and C) in the absence (control) or presence of miconazole (2.5 mg/kg) and of miconazole and L-NAME (10 mg/kg) and (B and D) in the absence or presence of 14,15-EEZE (2.5 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is significantly different from basal. Each value represents the mean ± standard error of the mean for n = 7 to 11 rats. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control. Effect of ACTH on ABF We also studied the effect of ACTH on ABF. Intravenous injection of ACTH increased ABF at all dosages tested (maximal increase = 91.4 ± 10.8 PU with 1000 ng ACTH/kg) (Fig. 4A). ACTH did not alter MAP (Fig. 4B). The time course of ABF responses to ACTH (1 ng/kg) shows that ABF increased and plateaued in 1.0 to 1.7 minutes and returned to basal levels within 4 to 5 minutes (Fig. 4C). The injection of saline (0.1 mL) alone did not alter ABF. The ACTH-induced increase in ABF was not affected by L-NAME (10 mg/kg) pretreatment (Fig. 4A) or by indomethacin (data not shown). The increase in ABF was decreased by pretreatment with the CYP inhibitor miconazole (2 mg/kg) from 91.4 ± 4.7 PU to 66.0 ± 8.9 PU (Fig. 5A). However, this decrease in ABF with miconazole was not further changed with the combination of miconazole and L-NAME (Fig. 5A). Miconazole alone did not alter ABF. Pretreatment of the rat with 14,15-EEZE (2.5 mg/kg) did not affect the ACTH increase in ABF at lower dosages (0.1 to 1 ng/kg) but eliminated the increase in ABF at higher dosages (10 to 1000 ng/kg) (Fig. 5B). Thus, PGs do not appear to contribute to the increase in ABF to Ang II and ACTH. However, the studies with L-NAME show that NO contributes to the ABF increase with Ang II but not with ACTH. CYP metabolites of arachidonic acid, the EETs, contribute to the increase in ABF with Ang II and ACTH. Both EETs and NO in combination mediate the ABF increase with Ang II. Figure 4. View largeDownload slide Effects of ACTH on (A) ABF and (B) MAP in the absence (control) and presence of L-NAME (10 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is significantly different from basal. (C) Time course of ABF changes in response to a bolus injection of ACTH (1 ng/kg). The 10-second mean values for change in ABF are indicated for the first 2 minutes, followed by the 60-second mean values up to 4 minutes. Each value represents the mean ± standard error of the mean for n = 5 to 10 rats. Figure 4. View largeDownload slide Effects of ACTH on (A) ABF and (B) MAP in the absence (control) and presence of L-NAME (10 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is significantly different from basal. (C) Time course of ABF changes in response to a bolus injection of ACTH (1 ng/kg). The 10-second mean values for change in ABF are indicated for the first 2 minutes, followed by the 60-second mean values up to 4 minutes. Each value represents the mean ± standard error of the mean for n = 5 to 10 rats. Figure 5. View largeDownload slide Effects of ACTH on (A and B) ABF and (C and D) MAP (A and C) in the absence (control) or presence of miconazole (2.5 mg/kg) and of miconazole and L-NAME (10 mg/kg) and (B and D) in the absence or presence of 14,15-EEZE (2.5 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is significantly different from basal. Each value represents the mean ± standard error of the mean for n = 7 to 11 rats. *P < 0.05, **P < 0.01 compared with control. Figure 5. View largeDownload slide Effects of ACTH on (A and B) ABF and (C and D) MAP (A and C) in the absence (control) or presence of miconazole (2.5 mg/kg) and of miconazole and L-NAME (10 mg/kg) and (B and D) in the absence or presence of 14,15-EEZE (2.5 mg/kg). Maximal changes in ABF and MAP are depicted. Effect on ABF is significantly different from basal. Each value represents the mean ± standard error of the mean for n = 7 to 11 rats. *P < 0.05, **P < 0.01 compared with control. Effect of inhibitors on plasma eicosanoids Plasma PGE2 and TxB2, 15-HETE and 14,15-EET and 11,12-EET, and DHET were used as indices of the activity of the cyclooxygenase, lipoxygenase and CYP epoxygenase pathways of arachidonic acid metabolism, respectively (Table 1). Compared with control, vehicle-treated rats, the plasma concentrations of TxB2 and PGE2 were decreased significantly with indomethacin. The plasma concentrations of 15-HETE, EETs, and DHETs were similar between vehicle- and indomethacin-treated rats. Miconazole significantly decreased the plasma concentrations of EETs and DHETs without changing 15-HETE. The plasma concentrations of TxB2 and PGE2 were decreased with miconazole; however, these decreases did not attain statistical significance. These data support the inhibition of cyclooxygenase by indomethacin and CYP epoxygenase by miconazole in the in vivo dosages used. Furthermore, the failure of indomethacin to alter the ABF changes with Ang II and ACTH occurred in the presence of documented cyclooxygenase inhibition by the drug. Thus, cyclooxygenase metabolites do not contribute to Ang II- and ACTH- mediated changes in ABF. These data also indicate that miconazole inhibits the ABF increases to Ang II and ACTH by suppressing CYP epoxygenase activity and EET and DHET synthesis. Though acting at different sites in the CYP-EET pathway, 14,15-EEZE and miconazole had similar effects. Table 1. Effect of Indomethacin and Miconazole Treatment on Plasma Concentrations of Selected Eicosanoids   Plasma Concentration (ng/mL)  Eicosanoid  Control  Indomethacin  Miconazole  TxB2  2.57 ± 1.52  0.00 ± 0.00a  1.52 ± 1.02  PGE2  0.05 ± 0.01  0.0015 ± 0.001b  0.024 ± 0.013  15-HETE  2.82 ± 0.60  2.77 ± 1.03  2.38 ± 0.90  11,12-DHET  0.55 ± 0.05  0.62 ± 0.03  0.07 ± 0.04c  14,15-DHET  0.52 ± 0.05  0.54 ± 0.05  0.21 ± 0.03c  11,12-EET  0.42 ± 0.06  0.48 ± 0.06  0.22 ± 0.03a  14,15-EET  0.84 ± 0.10  0.85 ± 0.23  0.41 ± 0.08a    Plasma Concentration (ng/mL)  Eicosanoid  Control  Indomethacin  Miconazole  TxB2  2.57 ± 1.52  0.00 ± 0.00a  1.52 ± 1.02  PGE2  0.05 ± 0.01  0.0015 ± 0.001b  0.024 ± 0.013  15-HETE  2.82 ± 0.60  2.77 ± 1.03  2.38 ± 0.90  11,12-DHET  0.55 ± 0.05  0.62 ± 0.03  0.07 ± 0.04c  14,15-DHET  0.52 ± 0.05  0.54 ± 0.05  0.21 ± 0.03c  11,12-EET  0.42 ± 0.06  0.48 ± 0.06  0.22 ± 0.03a  14,15-EET  0.84 ± 0.10  0.85 ± 0.23  0.41 ± 0.08a  Data are presented as means ± standard error of the mean of four experiments. Two-tailed Student t test was used for determining the significance of observed differences between experimental values, with P < 0.05 considered statistically significant. a P < 0.05. b P < 0.01. c P < 0.005. View Large Discussion ABF is critical to the supply of oxygen, nutrients, and cholesterol for steroidogenesis and delivery of aldosterone and cortisol to their target organs (1, 2, 7). The regulation of ABF by steroidogenic stimuli and the intra-adrenal mediators of adrenal vascular tone are poorly understood. We developed an ABF method using laser Doppler flowmetry in anesthetized rats and demonstrated that Ang II and ACTH increase ABF (26). Using this method, this investigation characterized the effects of Ang II and ACTH on ABF in vivo and extended the previous investigations by describing the endogenous mediators involved. Earlier studies described conflicting effects of Ang II on ABF that may be attributed to differences in methods, Ang II dosage, or species studied. For instance, Ang II increased ABF in anephric rats (38). However, Ang II in higher concentrations reduced ABF (38, 39). These decreases in ABF to Ang II were increased by pretreatment with L-NAME, indicating that endogenous NO limits Ang II constriction (39). Also, in dexamethasone-treated rabbits and sheep with cervical adrenal autotransplant, Ang II had no effect on ABF (40, 41). The effect of ACTH on ABF has also been investigated in experimental animals (1). Similarly, controversy exists concerning the dosage and timing of the ABF increase with ACTH. For instance, physiological concentrations of ACTH did not affect ABF, but concentrations above the physiological range increased ABF (18, 19). ACTH produced an immediate increase in flow in the intact perfused canine adrenal gland in one study (20), whereas in another study, a delayed dilation to ACTH was observed (42, 43). ACTH also increased ABF in perfused fetal sheep adrenal glands (21). Intra-adrenal mediators of the ABF responses to Ang II and ACTH were not investigated in detail previously. However, in in vitro studies, dilation to Ang II was mediated by NO and EETs, whereas dilation to ACTH was mediated by EETs only (25, 27, 44) (Fig. 6). We tested these possibilities in vivo by using inhibitors of PG, NO, and EET synthesis. Intravenous injection of both Ang II and ACTH dose-dependently increased ABF. Ang II also increases MAP, but ACTH did not. The cyclooxygenase inhibitor indomethacin, in a dosage that significantly decreased plasma concentration of PGE2 and TxB2, had no effect on the Ang II increase in ABF, so PGs do not contribute to the increase in ABF to Ang II. This finding is consistent with the findings of in vitro studies in isolated adrenal arteries and ZG cells that indomethacin did not affect ZG cell–mediated vasorelaxations to Ang II (25). Increase in ABF to Ang II was inhibited 60% with L-NAME pretreatment, but L-NAME did not affect the ABF increase to ACTH. L-NAME did not affect the increase in MAP to Ang II, so changes in MAP could not explain the inhibition of ABF. These findings are consistent with the findings of previous in vitro studies in adrenal arteries that endothelial NO regulates relaxation to Ang II but not ACTH (25, 27) (Fig. 6). In isolated adrenal arteries, ACTH induces relaxation only in the presence of ZG cells. These relaxations to ACTH were not affected by endothelium removal or NO synthase inhibition. Figure 6. View largeDownload slide Summary of the regulation of adrenal vascular tone and ABF by Ang II and ACTH. Ang II–mediated vasodilation and increase in ABF are mediated by endothelial NO and ZG cell EETs and DHETs, whereas ACTH is mediated by ZG cell EETs and DHETs only. Arg, arginine; AT2, Ang II type 2 receptor; AT?, Ang II receptor, not type 1 or 2; cGMP, cyclic guanosine monophosphate; EETr, EET receptor; Gs, guanine nucleotide binding protein αs; GTP, guanosine triphosphate; K+, potassium; MCR, melanocortin receptor; NOS, NO synthase; PL, phospholipid; sGC, soluble guanylyl cyclase. Figure 6. View largeDownload slide Summary of the regulation of adrenal vascular tone and ABF by Ang II and ACTH. Ang II–mediated vasodilation and increase in ABF are mediated by endothelial NO and ZG cell EETs and DHETs, whereas ACTH is mediated by ZG cell EETs and DHETs only. Arg, arginine; AT2, Ang II type 2 receptor; AT?, Ang II receptor, not type 1 or 2; cGMP, cyclic guanosine monophosphate; EETr, EET receptor; Gs, guanine nucleotide binding protein αs; GTP, guanosine triphosphate; K+, potassium; MCR, melanocortin receptor; NOS, NO synthase; PL, phospholipid; sGC, soluble guanylyl cyclase. CYP inhibitor miconazole and the EET antagonist 14,15-EEZE inhibit ABF increases to Ang II and ACTH in vivo. The inhibitors did not affect MAP, indicating that changes in MAP cannot explain the reduced ABF increases to Ang II or ACTH. Previous studies in isolated adrenal arteries showed that Ang II stimulates endothelial cell Ang type 2 receptors that mediate release of NO (27, 28) (Fig. 6). Additionally, Ang II activates ZG cells that release EET to induce relaxation (25). ACTH does not affect vascular tone in isolated adrenal arterioles (44) but induces relaxation in presence of ZG cells (11). CYP inhibitors (miconazole and SKF525a), high K+, iberiotoxin, and 14,15-EEZE attenuated the relaxations to ACTH in vitro (11). In a previous study, miconazole blocked the ABF increase to a single dose of Ang II and ACTH (26). In the current study, miconazole attenuated but did not block the ABF increases to a range of concentrations of Ang II and ACTH. The reasons for these differences in ABF inhibition by miconazole are not known. However, these studies with miconazole support a role for CYP metabolites of arachidonic acid in the regulation of ABF. This conclusion is supported by miconazole causing a significant decrease in the plasma concentrations of 14,15-EET, 11,12-EET, and DHET. Additionally, the decrease in ABF to Ang II and ACTH with 14,15-EEZE indicates that the CYP metabolites are arachidonic acid–derived EETs and that ZG cell–derived EETs mediate a portion of the relaxation to Ang II and relaxations to ACTH in vivo. The combination of L-NAME and miconazole completely eliminated the increase in ABF to Ang II; however, the effect of this combination on the increase in ABF to ACTH was similar to that of miconazole alone. These findings indicate that combined mediators, endothelial NO and ZG cell–derived EETs, are involved in the increase in ABF to Ang II; however, only ZG cell–derived EETs explain the increase in ABF to ACTH in both in vivo and in vitro (Fig. 6). In studies in isolated coronary arteries, the endothelium-dependent relaxations to bradykinin were mediated by NO and EETs; however, the CYP and EET component was not apparent until NO synthase was inhibited (45–47). In these arteries, NO reduced CYP activity, so inhibition of NO synthesis restored EET synthesis. Such an interaction was not observed in adrenal arteries or adrenal arteries and ZG cells with Ang II (25, 27, 28). Also, ABF increases with Ang II have both NO and EET components that act in parallel. This effect may be due to NO and EETs being synthesized by different cells in the adrenal gland, EETs by ZG cells and NO by endothelial cells (Fig. 6). Unlike endothelial cells, ZG cells do not contain NO synthase or synthesize NO, but both cell types synthesize EETs (11, 48, 49). Apparently, endothelial NO does not inhibit ZG cell EET synthesis, allowing the two mediators to act in parallel. Additionally, Ang II and bradykinin differ in their cellular actions and the endothelial mediators that are released. Bradykinin stimulates both NO and EET production from the coronary vascular endothelium (45, 46), whereas Ang II stimulates only NO production via endothelial Ang II receptor of adrenal arteries (25, 27, 28). The cellular pathways mediating these differences have not been described. Ang II causes vasoconstriction, whereas ACTH has no effects on vascular tone (11, 50). To maintain ABF in the presence of Ang II, endothelial NO is important to counteract the vasoconstriction and maintain or increase ABF (39). Ang II also acts on ZG cells to release EETs, promoting vasodilation and increases in ABF associated with stimulation of steroidogenesis. Because ACTH has no direct vascular effects (11), NO is not needed to oppose constriction, and only ZG cell–derived EETs are needed to link increases in ABF to steroidogenesis. In addition to EET release, Ang II and ACTH stimulate ZG cells to release aldosterone and corticosterone. Miconazole inhibits the synthesis of steroids and EETs (51, 52). However, steroids are not mediators of Ang II- or ACTH-induced dilation in vitro or ABF increases in vivo. Aldosterone and the glucocorticoid dexamethasone did not change vascular tone in vitro in isolated adrenal arteries (11). Also, 14,15-EEZE, like miconazole, attenuated the relaxations to Ang II and ACTH in vitro in isolated adrenal arteries as well as ABF increases in vivo, but unlike miconazole, 14,15-EEZE did not affect steroidogenesis (25). These studies indicate the importance of EETs and NO in the regulation of ABF by Ang II and EETs with ACTH in vivo (Fig. 6). From in vivo studies, it is not know with certainty whether the EETs are derived from ZG cells. However, in vitro studies indicate that ZG cells synthesize and release EETs in response to these stimuli. These studies indicate the importance of NO and EETs in increasing ABF with Ang II and EETs with ACTH and provide in vivo evidence that CYP metabolites of arachidonic acid, the EETs, mediate increases in ABF that are coupled to steroidogenesis. This ZG cell–EET pathway couples an increase in ABF with stimulation of steroidogenesis and promotes the increased delivery of oxygen, cholesterol, and nutrients for steroid synthesis and delivery of newly synthesized steroids to their target tissues. Abbreviations: 14,15-EEZE 14,15-epoxyeicosa-5(Z)-enoic acid 15-HETE 15-hydroxyeicosatetraenoic acid ABF adrenal blood flow ACTH adrenocorticotropic hormone Ang II angiotensin II CYP cytochrome P450 DHET dihydroxyeicosatrienoic acid EET epoxyeicosatrienoic acid L-NAME N-nitro-l-arginine methyl ester MAP mean arterial pressure NO nitric oxide PG prostaglandin PGE2 prostaglandin E2 PU perfusion unit TxB2 thromboxane B2 ZG zona glomerulosa. Acknowledgments The authors thank Gretchen Barg for her secretarial assistance. This research was conducted while Dr. Abdul Jabbar Shah was on sabbatical leave from COMSATS Institute of Information Technology, Abbottabad, Pakistan. Financial Support: These studies were supported by National Heart, Lung, and Blood Institute Grant HL-83297 (to W.B.C.); a Ralph and Marian Falk Medical Research Trust Bank of America, N.A., Trustee Grant (to J.R.F.); and Robert A. Welch Foundation Grant I-0011 (to J.R.F.). 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