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. 2010 Jul 28;151(10):4820–4829. doi: 10.1210/en.2009-1454

Urocortin 2 Lowers Blood Pressure and Reduces Plasma Catecholamine Levels in Mice with Hyperadrenergic Activity

Yusu Gu 1, Kuixing Zhang 1, Nilima Biswas 1, Ryan S Friese 1, Dennis H Lin 1, Sushil K Mahata 1, Masahiko Hoshijima 1, Daniel T O'Connor 1, Kirk L Peterson 1, Bhawanjit K Brar 1
PMCID: PMC2946150  PMID: 20668031

Abstract

Exaggerated adrenergic activity is associated with human hypertension. The peptide urocortin 2 (Ucn 2) inhibits catecholamine synthesis and secretion from adrenal chromaffin cells in vitro and administration to mammals lowers blood pressure (BP). The chromogranin A-null mouse (Chga−/−) manifests systemic hypertension because of excessive catecholamine secretion from the adrenal and decreased catecholamine storage. In the present study, we investigated whether systemic administration of Ucn 2 could reduce BP and adrenal and plasma levels of catecholamines in vivo. Ucn 2 peptide was administered to freely moving, conscious Chga−/− and wild-type control mice. Telemetry and HPLC measured changes in BP and catecholamine levels, respectively. In both groups of mice, Ucn 2 dose-dependently decreased BP, and this effect was mediated by corticotropin factor-receptor type 2. However, in Chga−/− mice, the maximal percentage decrease of systolic BP from basal systolic BP was 37% compared with only a 23% reduction in wild-type mice (P = 0.04). In Chga−/− mice only, Ucn 2 decreased adrenal and plasma levels of catecholamines as well as adrenal levels of tyrosine hydroxylase protein and phosphorylation. In vitro mechanistic studies demonstrated that Ucn 2 reduces both catecholamine secretion and tyrosine hydroxylase promoter activity, suggesting that the exaggerated action of Ucn 2 to reduce BP in the Chga−/− mouse is mediated through inhibition of both catecholamine synthesis and secretion. The data suggest that Ucn 2 may be therapeutically useful in regulating the exaggerated sympathoadrenal function of hyperadrenergic hypertension.

Urocortin 2 peptide lowers blood pressure in mice with high blood pressure, in part by decreasing adrenal production and release of catecholamines.

Urocortin 2 (Ucn 2), also known as stresscopin-related peptide, is a member of the mammalian corticotrophin-releasing factor family of peptides (1,2,3,4). Ucn 2 is coexpressed with its G protein-coupled receptor, corticotrophin-releasing factor receptor type 2 (CRFR2) in a number of tissues including the heart, vasculature, kidneys, gut, and skeletal muscle (5,6,7,8).

Both mRNA and protein for Ucn 2 are found in the human and rat adrenal medulla as well as PC12 pheochromocytoma cells (8). Exposure of human adrenal chromaffin cells to Ucn 2 suppresses catecholamine (CA) secretion by changes in subplasmalemmal actin filament polymerization, an effect that is blocked by CRFR2 antagonism. Ucn 2 also inhibits CA synthesis in human chromaffin cells by reducing mRNA and protein expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in CA biosynthesis (8). Levels of circulating Ucn 2 in human plasma are unknown. However, the peptide (or synthetic analogs) could have a potential application for the treatment of hypertension because its administration to humans and animals causes vasodilation and blood pressure (BP) reduction (9,10,11,12,13,14,15,16).

Chromogranin A is required for the formation of CA secretory vesicles in chromaffin cells, and its expression is sufficient to induce a regulated secretory system in nonsecretory cells (17). Over the past 20 yr, phenotypic links between chromogranin A and essential (idiopathic, genetic) human and rodent hypertension have been observed repeatedly (18,19). Deletion of the chromogranin A gene (Chga−/−) in mice increases BP and plasma CA levels (20). Evidence suggests that Chga−/− mice have elevated plasma and decreased adrenal CA levels because removal of chromogranin A from the dense core secretory vesicles results in dysregulated (poor, diminished) transmitter storage and release (excessive). Thus autonomic control of the circulation is altered, leading to hypertension (20). Hence, the Chga−/− mouse is a model of hyperadrenergic activity in essential hypertension. Although the knockout is etiologically a Mendelian/monofactorial model, the human disease, by contrast, is multifactorial (21,22).

In the present study, we hypothesized that Ucn 2 would reduce the elevated systolic BP (SBP) and diastolic BP (DBP) and normalize excessive plasma CA levels in the Chga−/− mouse because the peptide inhibits CA secretion from adrenal chromaffin cells and biosynthesis in vitro (8). Specifically, we compared the effect of systemic administration of Ucn 2 peptide on BP reduction in freely moving Chga−/− and wild-type (WT) control mice. The action of the peptide on adrenal and plasma epinephrine (EP) and norepinephrine (NE) levels and adrenal TH protein expression and activity, assayed by phosphorylation of the enzyme at Ser-40, were also determined (23). The results indicate that Ucn 2 disproportionately reduces both SBP and DBP in hyperadrenergic mice, a potential therapeutic effect resulting from reduction of both adrenal and plasma levels of CAs and suppressed production of adrenal TH.

Materials and Methods

Animals

All procedures were carried out in accordance with the guidelines set by the University of California, San Diego, Institutional Animal Care Program and with Institutional Animal Care and Use Committee approval and by National Institutes of Health guidelines. Chga−/− and WT mice have been previously reported (20). In this study the same mice were used for BP, CA, and TH analysis, with additional mice for CA determination.

Telemetry

BP was measured using the Data Sciences International (Transoma Medical, St. paul, MN) PhysioTel telemetry system. Adult male mice (5–6 months old) with body weights of approximately 30 g were anesthetized with isoflurane (5% for induction and 2% for maintenance), and a catheter was implanted in the left carotid artery coupled to a TA11PA-C10 (Data Sciences International) transmitter as before (20). Telemetry signals were received by an antenna below the cage that relayed the data to a signal processor (DataQuest A.R.T. Gold, version 2.3; Data Sciences International) connected to a desktop personal computer (Hewlett-Packard, Portland, OR). Ten days were allowed for normalization of the diurnal pattern of BP after the implantation surgery before recording BP. Baseline BP and heart rate [HR; beats/min (BPM)] were determined by averaging 10 consecutive seconds of data every 15 min over 20 h.

Drug treatments

Synthetic mouse Ucn 2 peptide and the CRFR2 antagonist astressin 2B (Ast-2B) (24), obtained from Jean Rivier (Salk Institute, La Jolla, CA) were dissolved to a concentration of 1 mg/ml in double-distilled H2O containing 0.1% BSA (pH 7.4), aliquoted, and stored at −70 C until use. For injection, Ast-2B or the peptide was dissolved in an appropriate volume of saline and administered as a single ip bolus of 100 μl. Drug treatments were performed on separate days. Chga−/− mice were injected with 100 μl of Ucn 2 (0.1, 1, or 7.5 μg) or saline. BP and HR were recorded at intervals of 15 min for 10 sec for up to 12 h after injection. Ast-2B (5 μg) or saline was administered as a single ip bolus of 100 μl in volume, 35 min before injection of Ucn 2 (1 μg). To determine whether Ucn 2 directly increases HR independent from the reduction of BP and a reflex effect of the sympathetic nervous system, the β-1- and β-2-adrenergic receptor antagonist, esmolol (150 μg/kg · min) was administered for 30 min as an iv infusion to anesthetized, micromanometer-catheter cannulated WT mice as before (14). Ucn 2 (5 μg) was then given as an iv bolus, and HR was recorded for 10 min using WINDAQ (Dataq Instruments, Akron, OH).

Adrenal and plasma CA analysis

Thirty minutes after an injection with Ucn 2 (1 μg) or saline, the same mice used for BP analysis were terminally anesthetized with sodium pentobarbitone (50 mg/kg, ip). Approximately 0.5 ml of blood was withdrawn from the heart of each mouse by cardiac puncture using heparin (1000 U/ml; Abraxis Pharmaceutical Products, Schaumburg, IL)-coated 1-ml Luer-Lok disposable syringes and 25-gauge needles (Becton Dickinson, Lincoln Park, NJ) via a single puncture in the chamber of the left ventricle. Blood was withdrawn slowly to prevent the heart from collapsing. Blood was transferred into heparin-coated 1.5-ml tubes (Axygen, Union City, CA) and immediately centrifuged at 10,000 rpm to separate the plasma, which was transferred into a new tube. The plasma and adrenals were snap frozen under liquid N2. Plasma CAs in 0.2 ml of plasma were measured using a previously reversed phase-HPLC-electrochemical detection protocol (25). The right adrenal gland was homogenized in 0.3 ml PBS and sampled for protein estimation. Then 0.25 ml of perchloric acid (0.8 n) was added to 0.25 ml of the homogenate and centrifuged at 14,000 rpm for 10 min at 4 C. The supernatant was removed into a new tube, and 3,4-dihydroxybenzylamine with sodium metabisulfite was added to achieve final concentrations of 10 ng and 0.125 mm, respectively. Alumina (40 mg) was then added and the pH increased by the addition of 0.4 ml of Tris buffer (pH 8.6). After 20 min and washing twice with water, CAs were eluted with 0.2 ml of 0.1 n HCl/0.125 mm sodium metabisulfite.

CA levels were determined using HPLC coupled to an electrochemical detector (Waters 600E multisolvent delivery system and Waters 2465 electrochemical detector; Waters, Milford, MA). Separation was performed on an Atlantis dC18 column (2.1 × 150 mm, 3 μm) (Waters). The mobile phase used was a mixture composed of phosphate-citrate buffer [2 mm NaH2PO4, 268 μm Na2-EDTA, 50 mm sodium citrate, 10 mm diethylamine hydrochloride, 0.072% 1-octanesulfonic acid (pH is adjusted to pH 3.1, using phosphoric acid), 2.2% N, N-dimethylacetamide)/acetonitrile at 95:5 (vol/vol)]. A flow rate of 0.25 ml/min was used with isocratic mobile phase. The electrode potential was set at +0.6 V. A standard stock solution of NE, EP, and 3,4-dihydroxybenzylamine was diluted with 0.1 n HCl/0.125 mm sodium metabisulfite solution. For plasma and adrenal CA, 500 and 10 pg of standard NE and EP were used, respectively. Data were analyzed using Empower software (Waters) and CA levels normalized according to the recovery of internal standard (25). Plasma and adrenal CA levels were expressed as picograms per milliliter and micrograms per milligram protein, respectively.

Adrenal TH levels

Thirty minutes after injecting Ucn 2 or saline, the left adrenal gland from each mouse was individually homogenized in 0.5 ml of ice-cold 0.2 m sucrose, Tris maleate (10 mm, pH 7.0) buffer, supplemented with 2 mm EDTA, 1 mm sodium orthovanadate, 10 mm sodium pyrophosphate, and protease inhibitor cocktail (Sigma, St. Louis, MO). Protein content in the cytosolic fractions was determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA). Twenty micrograms of individual cytosolic protein extract were subjected to Western immunoblot analysis, and all samples were run on the same gel. Each lane corresponds to a single mouse adrenal extract. A single nitrocellulose membrane was probed for phosphorylated TH at Ser-40 (P-TH) (P-TH antibody, catalog no. T9573; Sigma). The membrane was then stripped with 0.2 m glycine (pH 2.7) for 30 min before washing three times for 10 min with PBS and blocking for 1 h in 10% (wt/vol) nonfat milk in PBS. The membrane was then probed for total TH (anti-TH; Santa Cruz Biotechnologies, Santa Cruz, CA), and after stripping, probed for actin (anti-C-19, Santa Cruz). The P-TH antibody is specific for phosphorylated TH at residue Ser-40 (26).

CRFR2 gene expression studies

Age-matched adult (∼12 weeks old), male Chga−/− mice and WT control mice were obtained from the local breeding colony as before (27). Total RNA was extracted from isolated adrenal glands in WT (n = 3) and Chga−/− (n = 3) mice by the RNAzol (guanidinium thiocyanate) kit (TelTest, Friendswood, TX) followed by ribonuclease-free deoxyribonuclease I (QIAGEN, Valencia, CA) treatment to eliminate residual genomic DNA. Adrenal gene (mRNA) expression was measured using standard Affymetrix protocols and Affymetrix (Santa Clara, CA) MG-U74Av2 GeneChips. Statistical analysis of microarray data were performed using a Bayesian statistical method known as variance-modeled posterior inference with regional exponentials (VAMPIRE) (27).

Statistics

Raw basal values of BP and HR, presented as mean ± sem, were analyzed using the Student t test. The effect of drugs on raw BP and HR data were examined using the general linear model. In the model, treatment and time were treated as fixed factors. Post hoc Bonferroni test was examined using the least significance difference (LSD) correction to avoid multiplicity errors at specific BP measurements compared with saline control-treated mice. All data were analyzed within SPSS version 13 (SPSS Inc., Chicago, IL) with the significance level set at P < 0.05. Data are presented as mean ± sem. The maximum percentage decrease of BP from basal values for each mouse was calculated to compare normalized BP reductions. The percent reduction of BP was averaged for each mouse type, and the Student t test was used to analyze differences. For protein analysis, TH and P-TH homogenates were quantified using Image J software (National Institutes of Health, Bethesda, MD), normalized to actin, and fold increase calculated from the WT saline-treated mice. Two-way ANOVAs were conducted on P-TH and TH protein studies (StatView 5.0, SAS Institute, Cary, NC), followed by t test for comparisons within the same genotype and saline and Ucn 2 treatment. For plasma and adrenal CA levels in WT and Chga−/−, or other traits not normally distributed, the nonparametric Mann Whitney U test [(asymptotic significance (two tailed)] was used to determine the effects of Ucn 2.

Results

Ucn 2 decreases BP in WT an Chga−/− hypertensive mice

Chga−/− mice have elevated basal SBP (WT = 113 ± 3.8 mm Hg vs. Chga−/− = 124 ± 3.54 mm Hg, *, P = 0.05, n = 7, increase of 11 mm Hg) and DBP (WT = 88.8 ± 3.4 mm Hg vs. Chga−/− = 105.7 ± 2.3 mmHg, *, P = 0.001, n = 7, increase of 16 mm Hg) compared with their WT littermate controls (Fig. 1, A and B). A representative 1-sec trace recording of WT and Chga−/− BP before injection is shown (Fig. 1C). Administration of 0.1 μg of Ucn 2 peptide to WT mice did not reduce SBP and DBP (Fig. 2, A and B). In Chga−/− mice, 0.1 μg of Ucn 2 decreased SBP or DBP at 15, 30, and 60 min after injection (Fig. 2, D and E). One microgram of Ucn 2 decreased SBP and DBP in both WT and Chga−/− mice compared with saline control. Injection of 7.5 μg of Ucn 2 to these mice failed to further decrease BP in either strain compared with 1 μg of the peptide (data not shown).

Figure 1.

Figure 1

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Basal BP recordings in WT and Chga−/− mice. The mean basal (±sem) SBP and DBP of WT (A) and Chga−/− mice (B) are shown. Student’s t test determined BP differences between groups. Basal SBP was lower in WT (n = 7) vs. Chga−/− mice (n = 7) (*, P = 0.05). Basal DBP was lower in WT (n = 7) vs. Chga−/− mice (n = 7) (*, P = 0.001). Representative 10-sec trace recordings of both mouse strains types are shown (C).

Figure 2.

Figure 2

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Reduction of BP in WT and Chga−/− mice by Ucn 2. Ucn 2 was administered as an ip bolus to WT and Chga−/− mice at doses of 0.1 and 1 μg. The effects of 0.1 and 1 μg of Ucn 2 on the SBP in WT (A) and Chga−/− (D) mice, DBP in WT (B) and Chga−/− (E), and HR in WT (C) and Chga−/− mice (F) are shown. Raw BP and HR data were examined using a general linear model with treatment and time as fixed factors. A post hoc Bonferroni test was examined using the LSD correction. Data were analyzed within SPSS version 13 (SPSS) with the significance level set at P < 0.05 (#, 0.1 μg; *, 0.1 μg) compared with saline injected mice at the same time point. Data points represent mean ± sem of seven mice per group. In WT mice (A) for SBP, the statistics were as follows: overall, F = 5.7, P < 0.001; time, F = 3.49, P < 0.001; treatment, F = 116.3, P < 0.001; treatment by time, F = 1.7, P = 0.007. In WT mice for DBP (B) the statistics were as follows: overall, F = 7.34, P < 0.001; time, F = 157, P < 0.001; treatment, F = 3.77, P < 0.001; treatment by time, F = 2.35, P < 0.001. In Chga−/− mice for SBP (D), the statistics were as follows: overall, F = 5.47, P < 0.001; time, F = 19.8, P < 0.001; treatment, F = 8.0, P < 0.001; treatment by time, F = 2.67, P < 0.001. In Chga−/− mice for DBP (E), the statistics were as follows: overall, F = 5.0, P < 0.001; time, F = 21.6, P < 0.001; treatment, F = 7.12, P < 0.001; treatment by time, F = 2.3, P = 0.002. Both SBP and DBP in Chga−/− mice were reduced at the 15-, 30-, and 60-min time points in mice treated with 0.1 μg Ucn 2 (#, P < 0.05). In WT mice (C) for HR, the statistics were as follows: overall, F = 1.54, P = 0.009; time, F = 1.95, P = 0.005; treatment, F = 5.03, P = 0.007; treatment by time, F = 0.804, P = 0.78. In Chga−/− mice (F) for HR, the statistics were as follows: overall, F = 1.80, P = 0.008; time, F = 1.961, P = 0.039; treatment, F = 6.49, P = 0.002; treatment by time, F = 1.26, P = 0.213. Compared with saline, 1 μg Ucn 2 increased HR at 60 min in WT mice (*, P < 0.05) (C). In Chga−/− mice, compared with saline, 0.1 μg Ucn 2 increased HR at 45, 75, 90, and 105 min after injection (F), and 1 μg Ucn 2 increased HR at 90 and 105 min after injection (*, P < 0.05).

BP-lowering effect of Ucn 2 is more pronounced in Chga−/− mice

To account for differences in basal BP across strains, we expressed the maximum decrease in BP as a percent change from the average basal values. The maximum percentage decrease of SBP in WT mice was 23.1 ± 4.9% after injection with 1 μg of peptide and is less than the 37.4 ± 6.3% decrease of SBP in Chga−/− mice (P = 0.04). For DBP the maximum decrease in WT mice from basal was 23.1 ± 4.9%, a value less than the 38.5 ± 6.6% decrease in DBP in Chga−/− mice (P = 0.04). The average decrease in absolute values of SBP from basal was 21.3 ± 4.5 mm Hg in WT mice vs. 47.1 ± 9.0 mm Hg in Chga−/− mice (*, P = 0.025), whereas the decrease in DBP was 22.9 ± 3.7 mm Hg in WT mice vs. 44.3 ± 9.8 mm Hg in Chga−/− mice (*, P = 0.03).

Action of Ucn 2 on HR

In both strains, basal 24-h HR values were similar (WT = 552.1 ± 24.9 BPM vs. Chga−/− = 552.4 ± 14.6 BPM). Compared with saline, in WT mice, 0.1 μg Ucn 2 had no effect on HR; however, 1 μg Ucn 2 increased HR only at 60 min (*, P < 0.05) (Fig. 2C). In Chga−/− mice given 0.1 μg of Ucn 2, HR increased at 45, 75, 90, and 105 min after injection (#, P < 0.05) compared with saline administration (Fig. 2F), whereas 1 μg Ucn 2 increased HR at 90 and 105 min after injection (*, P < 0.05). In anesthetized, micromanometer-catheter cannulated WT mice (n = 3), in the presence of esmolol, Ucn 2 administration increased HR from 378.3 ± 46.9 BPM (before Ucn 2) to 568 ± 75.74 BPM (after Ucn 2), P < 0.05.

The BP-lowering effect of Ucn 2 is mediated by CRFR2

To determine whether the Ucn 2-mediated BP-lowering effect was CRFR2 mediated, Ast-2B was administered before saline or Ucn 2. Ast-2B alone had no effect on SBP or DBP compared with saline (data not shown). In the absence of Ast-2B, in WT and Chga−/− mice, Ucn 2 reduced SBP (Fig. 3, A and C) and DBP (Fig. 3, B and D) compared with saline-treated mice (*, P < 0.05). There were no differences in BP between saline-treated mice and the Ast-2B + Ucn 2-treated mice, with the exception of the 60-min time point after injection of Ucn 2 in Chga−/− mice, in which Ucn 2 reduced DBP compared with saline (#, P < 0.05).

Figure 3.

Figure 3

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The BP-lowering effect of 1 μg of Ucn 2 is inhibited after a 30-min pretreatment with 5 μg of CRFR2 antagonist Ast-2B. Ast-2B was injected ip 30 min before an iv bolus injection of 1 μg of Ucn 2 (arrow shows injection of Ucn 2). The effects of drugs on raw BP data were examined using the general linear model. In the model, treatment and time were treated as fixed factors. A post hoc Bonferroni test was examined using the LSD correction. Data were analyzed within SPSS version 13 (SPSS) with the significance level set at P < 0.05. Data points represent mean ± se of seven mice per group. In the absence of Ast-2B, Ucn 2 reduced SBP (A) and DBP (B) for up to 90 min compared with saline-treated mice (P < 0.05) (SBP: overall, F = 6.8, P < 0.001; time, F = 3.8, P < 0.001; treatment, F = 117, P < 0.001; treatment by time, F = 1.54, P = 0.045; for DBP: overall, F = 8.1, P < 0.001; time, F = 3.8, P < 0.001; treatment, F = 140, P < 0.001; treatment by time, F = 1.95, P = 0.003). A similar effect was observed in Chga−/− mice wherein Ucn 2 reduced SBP (C) and DBP (D) at all time points (SBP: overall, F = 7.5, P < 0.001; time, F = 9.3, P < 0.001; treatment, F = 37.3, P < 0.001; treatment by time, F = 2.9, P = 0.002; for DBP: overall, F = 6.0, P < 0.001; time, F = 11.2, P < 0.001; treatment, F = 25.9, P < 0.001; treatment by time, F = 0.97, P = 0.49). There was no difference in BP between the saline-treated and the Ast-2B + Ucn 2-treated Chga−/− mice, with the exception of DBP 60 min after injection of Ucn 2 (P < 0.05).

Ucn 2 decreases plasma and adrenal CA content as well as CA secretion from chromaffin cells in Chga−/− mice

Maximal BP reduction by Ucn 2 occurred between 30 and 45 min after injection; therefore, mice were injected with saline or 1 μg of Ucn 2, and 30 min after the injection, blood was drawn and the adrenals extracted. As previously reported (20), Chga−/− mice exhibited elevated plasma levels of NE and EP compared with WT mice (WT: NE = 4060.2 ± 960.9 pg/ml, EP = 712.4 ± 172.9 pg/ml vs. Chga−/−: NE = 7090.4 ± 849.3 pg/ml, EP = 1270 ± 192.7 pg/ml, *, P = 0.046 for NE, *, P = 0.05 for EP, Fig. 4A). As previously reported (20), Chga−/− mice exhibit lower adrenal CA levels compared with WT mice (*, P = 0.035 for NE, *, P = 0.047 for EP, Fig. 4B). Administration of Ucn 2 reduced elevated plasma levels of NE and EP in Chga−/− mice toward levels comparable with WT mice (WT mice + saline; NE = 4060 ± 960.9 pg/ml, EP = 712.4 ± 172. 9 pg/ml vs. Chga−/− + Ucn 2: NE = 4801 ± 807.4 pg/ml, EP = 806.6 ± 439.0 pg/ml; #, P = 0.045 for NE, **, P = 0.0032 for EP). Ucn 2 treatment also reduced adrenal NE (#, P = 0.006) and EP (**, P = 0.015) in Chga−/− mice, whereas saline had no effect. In cultured PC12 chromaffin cells, Ucn 2 substantially inhibited secretagogue (membrane depolarization by KCl) stimulated NE secretion (Supplemental Fig. 1 Journals Online web site at http://endo.endojournals.org) (KCl vs. KCl + Ucn 2, *, P = 1.5 × 10−5).

Figure 4.

Figure 4

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Ucn 2 reduces the plasma and adrenal CA levels in WT and Chga−/− mice. The effect of ip administered Ucn 2 (1 μg) or saline on plasma (A) and adrenal catecholamine (B) levels in WT (n = 6 for saline, n = 8 for Ucn 2) and Chga−/− mice 30 min after the injection was determined (n = 6 for saline, n = 9 for Ucn 2). For plasma and adrenal CA levels in WT and Chga−/− mice, the nonparametric Mann Whitney U test was used to determine the effect of Ucn 2 (SPSS version 13; SPSS). Error bars represent mean ± sem. A, Plasma NE levels were lower in WT + saline vs. Chga−/− + saline mice (*, P = 0.046). Ucn 2-treated Chga−/− mice had lower NE levels (Chga−/− + Ucn 2 vs. Chga−/− + saline; #, P = 0.045). Similarly, the plasma EP levels were higher in Chga−/− mice (*, P = 0.05) compared with WT mice, and Ucn 2 reduced EP levels in Chga−/− mice only (Chga−/− + Ucn 2 vs. Chga−/− + saline; **, P = 0.0032). B, For adrenal CA levels, NE levels were greater in the WT + saline vs. Chga−/− + saline mice (*, P = 0.035). Ucn 2-treated Chga−/− mice had lower NE levels (Chga−/− + Ucn 2 vs. Chga−/− + saline; #, P = 0.006). Adrenal EP levels were higher in WT + saline-treated mice compared with Chga−/− + saline-treated mice (*, P = 0.047). EP levels were reduced in Chga−/− mice administered with Ucn 2 (Chga−/− + Ucn 2 vs. Chga−/− + saline; **, P = 0.015).

Ucn 2 decreases TH and P-TH levels in the adrenals of Chga−/− mice

Because Ucn 2 decreased adrenal and plasma CA levels [similar to its effects in vitro (8)], we show that in Chga−/− mice only, Ucn 2 reduced total P-TH and TH levels [(P-TH-genotype-F = 0.04, P = 0.85; treatment-F = 0.41, P = 0.53; genotype × treatment-F = 20.55, P = 0.001) (TH-genotype-F = 0.01, P = 0.9; treatment-F = 2.06, P = 0.18; genotype × treatment-F = 21.53, P = 0.001)]. Ucn 2 resulted in decreased P-TH and TH levels in Chga−/− mice only (*, P = 0.003 for P-TH and *, P = 0.01 for TH) (Fig. 5, A and B). P-TH levels normalized to TH remained linear (WT saline vs. WT Ucn 2-P = 0.18, Chga−/− saline vs. Chga−/− + Ucn 2, P = 0.27) (Fig. 5C). In vitro TH promoter studies showed that Ucn 2 causes a dose-dependent decline in TH promoter activity (Supplemental Fig. 2).

Figure 5.

Figure 5

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Ucn 2 reduces TH levels in Chga−/− mice only. The effect of ip administered Ucn 2 (1 μg) or saline on adrenal P-TH (A) and TH (B) protein levels normalized to actin and then expressed as mean fold compared with the saline-treated WT mice is shown. Error bars represent mean ± sem. A, Two-way ANOVAs were conducted on P-TH protein studies (StatView 5.0). Ucn 2 treatment reduced P-TH levels in Chga−/− mice (P-TH-genotype, F = 0.04, P = 0.85; treatment, F = 0.41, P = 0.53; genotype × treatment, F = 20.55, P = 0.001). A t test showed that Ucn 2 significantly decreased P-TH levels in Chga−/− mice only (*, P = 0.003). B, Ucn 2 reduced TH levels in Chga−/− mice (TH genotype, F = 0.01, P = 0.9; treatment, F = 2.06, P = 0.18; genotype × treatment, F = 21.53, P = 0.001). A t test showed that Ucn 2 decreased TH levels in Chga−/− mice (*, P = 0.01). C, The P-TH/TH levels remained similar (WT saline vs. WT Ucn 2, P = 0.18, Chga−/− saline vs. Chga−/− + Ucn 2, P = 0.27).

CRFR2 gene expression

Global adrenal gene (mRNA) expression of CRFR2 was measured using MG-U74Av2 GeneChips (Affymetrix). Basal expression levels of CRFR2 were similar in WT and Chga−/− mice (Affymetrix probe set ID 162152_r_at: WT = 273.3, Chga−/− = 213.5, P value for Chga−/− mice vs. WT mice = 0.35).

Discussion

Overview

We show for the first time that Ucn 2 causes an exaggerated fall in both SBP and DBP in freely moving, conscious, hyperadrenergic Chga−/− mice. Whereas the BP-lowering effect of Ucn 2 in freely moving normal rats (11,13,28), anesthetized WT mice, and those with heart failure (14) has been described, such observations in freely moving hypertensive mice are heretofore lacking.

Because hypertension is defined by elevation of SBP or DBP, both parameters were measured in both mouse strains. As shown before (20), Chga−/− mice have elevated SBP and DBP compared with WT controls (Fig. 1, A–C). In both WT and Chga−/− mice, we observed significant BP lowering by Ucn 2 with a maximum effect between 30 and 45 min after injection for both DBP and SBP (Fig. 2, A and B and D–E). This time course of action of Ucn 2 is comparable with that demonstrated in conscious rats (11,13,28), sheep (12), and humans (29). The maximum reduction in BP from basal values induced by Ucn 2 was greater in Chga−/− mice compared with WT mice, suggesting that Ucn 2 acts on an additional pathway to further lower BP in Chga−/− mice. To probe the mechanism of the exaggerated effect of Ucn 2 on BP in Chga−/− mice, we focused on the cause of hypertension in this model: hyperadrenergic activity. The increase in BP in the Chga−/− mouse is ultimately caused by removal of chromogranin A from the dense core secretory vesicles, resulting in impaired transmitter storage and thus excessive release of CAs, altering autonomic control of the circulation, eventuating in hypertension (20,30).

Although Chga−/− mice have elevated plasma and decreased adrenal levels of neuropeptide Y as well as increased adrenal levels of corticosterone (20), our main focus was the action of Ucn 2 on NE and EP levels because Ucn 2 has been shown to inhibit CA synthesis and secretion from adrenal chromaffin cells in vitro, an effect antagonized by CRFR2 antagonism (8). Ucn 2 normalized elevated plasma CAs in Chga−/− mice at 30 min when BP reduction was maximal (Fig. 4A). Such short-term decrement in CA release by Ucn 2 may be explained by inhibition of secretion of previously synthesized/stored CA (20). An additional experiment in PC12 cells demonstrated that Ucn 2 inhibits secretagogue-stimulated NE secretion (Supplemental Fig. 1). The inhibitory effect of Ucn 2 on CA secretion has been demonstrated in PC12, primary rat and human adrenal chromaffin cells (8). Dermitzaki et al. (8) attributed inhibition of CA secretion to changes in subplasmaliminal actin filament polymerization. However, our data suggest that Ucn 2 inhibits KCl depolarization stimulated secretion of CAs.

In addition to decreasing plasma CA levels in Chga−/− mice, adrenal CA levels decreased after Ucn 2 treatment, suggesting that the peptide inhibits CA synthesis. This is highlighted by the selective action of Ucn 2 in Chga−/− mice reducing adrenal CA content (Fig. 4B) and adrenal P-TH and TH levels, thereby contributing to ongoing suppression of CA secretion (Fig. 5, A and B). We suggest that Ucn 2 (8) decreases TH promoter activity (Supplemental Fig. 2). Others have shown that Ucn 2 decreases TH RNA transcripts and protein in adrenal chromaffin cells (8); however, the effects of Ucn 2 on TH levels in vivo have not been demonstrated. The corresponding decrease in P-TH by Ucn 2 in Chga−/− mice may be secondary to the overall decline of TH protein (Fig. 5C). Because the present study was conducted in vivo, the direct mechanism by which Ucn 2 rapidly decreases adrenal TH protein within 30 min after Ucn 2 injection cannot be confirmed but may be attributed to transcriptional or posttranscriptional mechanisms such as ubiquitination/degradation (31,32). It is thought that in general, maximal changes in TH mRNA and protein content occur at 6–8 and 24 h after stressor exposure, respectively (32). However, the expression of many proteins involved in stress activated pathways such as heat shock proteins can be up- or down-regulated within minutes of sensing a stress, and this has been reported for the adrenal. For example, in response to surgical stress, specific and simultaneous heat shock protein induction occurs within 30 min in the adrenal cortex (33). Also, hypoxia-inducible transcription factors regulate heat shock protein expression in response to hypoxia in a number of cell types and have recently been shown to regulate the expression of TH in PC12 cells (34), suggesting that in specific stressful situations, in vivo TH protein may be more rapidly transcribed. With regard to the ubiquitination hypothesis, it is shown that the integral TH of the bovine adrenal chromaffin granule membrane is ubiquitinated, most likely monoubiquitinated and in the presence of an ATP-regenerating system. In that study the half-life of full-length TH was reported as 2.1 h, and a decrease in TH protein levels were observed after 1 h (31). Others have shown that the N terminus of TH controls protein stability through 14-3-3η proteins in PC12D rat pheochromocytoma cells (35). Whether Ucn 2 promotes TH ubiqitination or regulates 14-3-3η expression is not known.

The potent BP-lowering action of Ucn 2 is evident in both WT and Chga−/− mice. Then again, in Chga−/− mice, the maximal percentage decrease of systolic BP from basal systolic BP was 37% compared with only a 23% reduction in WT mice. Acute Ucn 2 administration was associated with a decrease of both adrenal and plasma levels of CAs when BP reduction was maximal in Chga−/− mice only. This decrease in CAs and BP was concordant with observed reductions in adrenal total TH and P-TH protein. The more exaggerated reduction of BP by Ucn 2 in the Chga−/− mouse is likely via inhibition of both CA synthesis and secretion.

Context and previous literature

Other studies have shown that decreasing TH levels or activity reduces sympathoadrenal activity and BP (36,37,38). For example TH antisense gene therapy causes hypotensive effects in spontaneously hypertensive rats, reducing adrenal TH levels, SBP, and EP and NE levels (38). Importantly, CRFR2 antagonism inhibits Ucn 2-mediated decreases in CA synthesis in adrenal chromaffin cells in vitro (8). This suggests that the Ucn 2 effects on CA levels in vivo are CRFR2 mediated. Decreased CA synthesis may be observed after chronic administration of the peptide. For example, chronic administration of Ucn 2 to spontaneously hypertensive rats (16) reduced cardiac hypertrophy, suggesting that the peptide reduces sympathetic overdrive associated with hypertension. Consistent with our findings that Ucn 2 lowers plasma CA levels, in the ovine rapid pacing model of heart failure, Ucn 2 lowers both BP and plasma EP (12). Also demonstrated in human subjects with heart failure, Ucn 2 lowers both BP and plasma NE (29). Although the effects of Ucn 2 on CA levels may be secondary to its salutary effects on cardiac performance, it is possible that the observed decrease in plasma CA levels reflects direct decrease in adrenal CA synthesis and release. Because Ucn 2 decreases plasma CA levels in Chga−/− mice, we would also expect the peptide to decrease HR (Fig. 2, C and F); however, in both WT and Chga−/− mice, Ucn 2 administration increased HR. Ucn 2 also increased HR in WT mice infused with esmolol and β-1- and β-2-adrenergic receptor knockout mice (data not shown), suggesting that the peptide directly increases HR independent of adrenergic receptors and the sympathetic nervous system.

Strengths and limitations

The data presented in this study show only one time point and dose of Ucn 2 (1 μg/mouse) for in vivo biochemical analysis, which were selected because the maximal decrease in BP occurred in mice injected under these conditions. Because the same mice used for telemetry studies were used for terminal CA and TH measurements, which require all blood and adrenal tissue, additional tissue and plasma measurements could not be made.

In dissecting the mechanisms underlying the Ucn 2-BP-lowering effect, we found that the BP-lowering effect of Ucn 2 was abrogated by pretreatment with the CRFR2 antagonist Ast-2B (Fig. 3). Importantly, CRFR2 mediates the effects of Ucn 2 to decrease CA secretion and synthesis in vitro (8). The role of CRFR2 in the Ucn 2-mediated BP decrease has been shown only in normotensive animals (10,13,14,28), but that is expected because Ucn 2 is a selective ligand for CRFR2. A number of signaling pathways downstream of CRFR2 have been implicated in the vasodilatory action of Ucn 2. For example, Ucn 2 has been reported to induce vasodilation in anesthetized pigs (10) and conscious rats via a nitric oxide (NO)-dependent pathway (39). In addition to the NO pathway, adenylate cyclase-activated protein kinase A (40,41,42,43,44), protein kinase G (42,45,46), and prostaglandins (39) have all been implicated in CRFR2-mediated hypotension. However, in the present animal model, inhibitors of NO synthesis, adenylyl and guanylyl cyclases and prostaglandins had no effect on the Ucn 2-mediated BP reduction in WT and Chga−/− mice (data not shown). We also examined the basal adrenal expression levels of CRFR2 in WT and Chga−/− mice because differences in adrenal CRFR2 expression could explain elevated CA levels in Chga−/− mice. However, adrenal expression of CRFR2 transcripts was similar in both strains. It is possible that the Chga−/− mouse has lower levels of Ucn 2 peptide. However, conventional peptide assays would not be able to accurately quantify the adrenal, cardiac, vascular, or plasma peptide levels that are relatively low compared with tissues such as skin or skeletal muscle (47).

We acknowledge the potential limitations of collecting plasma CAs under anesthesia. Although CA levels reported here are relatively high, the values are comparable with those reported previously in these mice (20), and blood was drawn under the same conditions in both mice types. Also, during in vivo studies on Chga−/− mice, we cannot prove that Ucn 2 directly inhibits CA secretion because adrenergic blockade or adrenalectomy would disrupt the hyperadrenergic phenotype in this model. Instead we turned to in vitro models to supplement out in vivo findings (Supplemental Figs. 1 and 2).

Conclusions and perspectives

To summarize, the present study has identified a new action of Ucn 2 to regulate plasma and adrenal CA levels in hyperadrenergic hypertension using a specific mouse model of the disease. We cannot fully understand why Ucn 2 acts to decrease CA and TH levels in the Chga−/− mouse; however, in vitro studies show that Ucn 2 is a regulator of CA secretion and synthesis (8). The present study demonstrates that the peptide regulates CA levels in a mouse model of hyperadrenergic activity, suggesting that it is a regulator of sympathoadrenal function in addition to its cardioprotective (48) and beneficial hemodynamic effects in human heart failure (14).

Supplementary Material

[Supplemental Data]
en.2009-1454_index.html (740B, html)

Footnotes

This work was supported by Grants R01 DA011311 and P01 HL58120 from the Department of Veterans Affairs, National Institutes of Health (to S.K.M. and D.T.O.) and the Foundation for Research (to K.L.P. and B.K.B.).

Disclosure Summary: Y.G., K.Z., N.B., D.H.L., S.K.M., M.H., D.T.O., K.L.P., and B.K.B. have nothing to declare.

First Published Online July 28, 2010

Abbreviations: Ast-2B, Astressin 2B; BP, blood pressure; BPM, beats/minute; CA, catecholamine; Chga−/−, chromogranin A-null mouse; CRFR2, corticotrophin-releasing factor receptor type 2; DBP, diastolic BP; EP, epinephrine; HR, heart rate; LSD, least significance difference; NE, norepinephrine; NO, nitric oxide; SBP, systolic BP; TH, tyrosine hydroxylase; Ucn 2, urocortin 2; WT, wild type.

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[Supplemental Data]
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