The glucocorticoid receptor is required for experimental adrenocorticotrophic hormone-induced hypertension in mice.

1. In the present study, we have (i) measured basal blood pressure by telemetry in wild-type (WT) and glucocorticoid receptor knockout (GRKO) mice; (ii) investigated whether or not adrenocorticotrophic hormone (ACTH) can induce hypertension in GRKO mice; and (iii) investigated the effect of mineralocortocoid receptor blockade on the cardiovascular physiology of GRKO mice. 2. Male WT and GRKO mice were treated with ACTH (2mg/kg per day s.c.) or spironolactone (100mg/kg per day s.c.) for 1-2weeks. Blood pressure (BP) was measured using a radiotelemetry system. Urinary Na:K, blood glucose concentrations and haematocrit were also measured during the treatment period. 3. Baseline systolic blood pressure (SBP) was higher in GRKO mice (126±4 mmHg, mean±SEM, n=11) than WT mice (114±2mmHg, n=10; P<0.05). There was no significant difference in baseline haematocrit, blood glucose and urine Na:K ratio in WT and GRKO mice. ACTH raised SBP in WT (135±8mmHg, n=8; P<0.05), but not in GRKO mice (113±9mmHg, n=6). Spironolactone treatment did not alter SBP. 4. Basal SBP was higher in GRKO than WT mice; ACTH raised blood pressure in WT, but not GRKO mice; and spironolactone did not alter BP in GRKO or WT mice. These data, together with a previous study showing both ACTH and corticosterone are increased in GRKO mice, show that the GR is required for the development of ACTH-induced hypertension in mice. Increased endogenous ACTH levels in this model might contribute to the increased basal SBP in GRKO mice, possibly through residual fragments of GR. Mineralocorticoid receptors do not appear to play a critical role in maintaining BP in glucocorticoid receptor deficient mice.

Antioxidant vitamins and adrenocorticotrophic hormone-induced hypertension in rats.

This study examined whether the anti-oxidants ascorbic acid, alpha- or gamma-tocopherol, could modify adrenocorticotrophic hormone (ACTH)-hypertension in Sprague-Dawley rats, a model associated with increased oxidative stress. Systolic blood pressure (SBP) was measured by the tail-cuff method. After four days of ascorbic acid (AA) (200 mg/kg/day drinking) or alpha-tocopherol (500 mg/kg/d i.p. or feed), rats were co-administered ACTH (0.2 mg/kg/day s.c.) or saline for 11 days (prevention studies). In reversal studies, ACTH/saline was administered for 15 days, and from day 9, alpha- or gamma-tocopherol (20 mg/kg/day) was added. ACTH increased SBP compared to saline (p < 0.05). AA or alpha-tocopherol failed to prevent and alpha- or gamma-tocopherol failed to reverse ACTH-induced hypertension. Thus, neither vitamin C (water soluble) nor E (lipid soluble) modified ACTH-induced hypertension in the rat.

Role of xanthine oxidase in dexamethasone-induced hypertension in rats.

1. Glucocorticoid-induced hypertension (GC-HT) in the rat is associated with nitric oxide-redox imbalance. 2. We studied the role of xanthine oxidase (XO), which is implicated in the production of reactive oxygen species, in dexamethasone-induced hypertension (dex-HT). 3. Thirty male Sprague-Dawley rats were divided randomly into four treatment groups: saline, dexamethasone (dex), allopurinol plus saline, and allopurinol plus dex. 4. Systolic blood pressures (SBP) and bodyweights were recorded each alternate day. Thymus weight was used as a marker of glucocorticoid activity, and serum urate to assess XO inhibition. 5. Dex increased SBP (110 +/- 2-126 +/- 3 mmHg; P < 0.001) and decreased thymus (P < 0.001) and bodyweights (P" < 0.01). Allopurinol decreased serum urate from 76 +/- 5 to 30 +/- 3 micromol/L (P < 0.001) in saline and from 84 +/- 13 to 28 +/- 2 micromol/L in dex-treated (P < 0.01) groups. 6. Allopurinol did not prevent dex-HT. This, together with our previous findings that allopurinol failed to prevent adrenocorticotrophic hormone induced hypertension, suggests that XO activity is not a major determinant of GC-HT in the rat.

Folic acid prevents and partially reverses glucocorticoid-induced hypertension in the rat.

BACKGROUND: To investigate the effect of folic acid on the increased pressure in rats treated with either adrenocorticotropic hormone (ACTH) or dexamethasone (Dex), and to further investigate the role of tetrahydrobiopterin (BH(4)) in any effect of folic acid by comparing the effect of BH(4) with that of folic acid in Dex hypertension. METHODS: Male Sprague-Dawley (SD) rats were treated with saline, subcutaneous ACTH (0.2 mg/kg/d) or Dex (10 microg/rat/d). Folic acid (0.04 g/L drinking) or BH(4) (10 mg/kg/d intraperitoneally) was started before (prevention) and during (reversal) glucocorticoid treatment. RESULTS: Saline, BH(4), vehicle for BH(4), or folic acid alone did not change systolic blood pressure (BP). Systolic BP was increased by ACTH and Dex. Folic acid, but not BH(4), prevented the development of hypertension caused by ACTH and Dex treatment. The ACTH and Dex hypertension were partially reversed by folic acid. The BH(4) increased plasma total biopterin concentrations. The Dex decreased plasma NOx concentrations but had no effect on plasma biopterin concentrations. The ACTH and Dex increased plasma F(2)-isoprostane concentrations and decreased serum homocysteine concentrations compared with control but had no effect on serum folate concentrations. Folic acid increased serum folate concentrations compared with control but had no effect on homocysteine concentrations. CONCLUSIONS: Folic acid prevented and partially reversed both ACTH and Dex hypertension in rats without modifying the increase in plasma F(2)-isoprostane concentrations. Given that BH(4) failed to prevent ACTH or Dex hypertension, folic acid is unlikely to be acting through increased BH(4) production. The precise mechanism for the BP-lowering effect of folic acid in this model of hypertension remains to be determined.

Apocynin but not L-arginine prevents and reverses dexamethasone-induced hypertension in the rat.

BACKGROUND: Dexamethasone (Dex)-hypertension in rats is associated with increased oxidative stress. We investigated effects of the NAD(P)H oxidase inhibitor apocynin and the nitric oxide (NO) precursor L-arginine on Dex-hypertension to determine the relative roles of NAD(P)H oxidase and uncoupling in the reactive oxygen species (ROS) generation and hypertension. METHODS: Male Sprague-Dawley rats (n = 10/group) received Dex (20 microg/kg/day subcutaneously) or saline (vehicle) for 14 days. In a prevention study, rats received 4 days of apocynin treatement (1.5 mmol/L in drinking water) followed by Dex/saline for 12 days. In reversal studies, apocynin or L-arginine was given from day 8 to 14. Systolic blood pressure (SBP) was measured by tail cuff, and thymus weight was used as a marker of glucocorticoid activity. RESULTS: Administration of Dex increased SBP (104 +/- 3 to 122 +/- 3 mm Hg, P < .01, mean +/- SEM) and decreased thymus and body weight (P' < .05). Apocynin alone had no effect on SBP, BW, or thymus weight. Apocynin prevented (122 +/- 4 Dex, 111 +/- 3 mm Hg Apocynin+Dex, P' < .05) and reversed Dex-hypertension (130 +/- 4 to 116 +/- 4 mm Hg, P < .01). L-arginine did not reverse Dex-hypertension. CONCLUSIONS: In male SD rats, apocynin but not l-arginine prevented and reversed Dex-hypertension, suggesting that NAD(P)H oxidase-mediated superoxide production but not endothelial nitric oxide synthase uncoupling is important in Dex-hypertension.

N-acetylcysteine antagonizes the development but does not reverse ACTH-induced hypertension in the rat.

We investigated the effect of antioxidant N-acetylcysteine (NAC) on adrenocorticotropic hormone (ACTH)-hypertension. Male Sprague-Dawley rats received NAC (10 mg/L) or water 4 days before ACTH/saline treatment for 13 days (prevention study). In a reversal study, NAC commenced on day 8 of ACTH/saline treatment and continued for 5 days. ACTH increased systolic blood pressure (SBP) in water drinking rats (111 +/- 1 to 131 +/- 3 mmHg, p < 0.001). In the prevention study, NAC + ACTH increased SBP (108 +/- 2 to 120 +/- 2 mmHg, p < 0.001) but less than ACTH alone (p' < 0.05). In the reversal study, NAC had no significant effect (132 +/- 4 to 124 +/- 3 mmHg, ns). Thus, NAC partially prevented but did not reverse ACTH-induced hypertension.

Apocynin but not allopurinol prevents and reverses adrenocorticotropic hormone-induced hypertension in the rat.

BACKGROUND: Adrenocorticotropic hormone (ACTH)-induced hypertension in the rat is accompanied by increased oxidative stress. This study examines the enzymatic source of reactive oxygen species in ACTH-hypertension. METHODS: Male Sprague-Dawley rats were divided into 10 groups of 10-20 rats per group. The NAD(P)H oxidase inhibitor apocynin (1.5 mmol/L in drinking water) or the xanthine oxidase inhibitor allopurinol (200 mg/kg/day via food) were administered daily. After 4 days, rats were co-administered ACTH (0.2 mg/kg/day) or saline by subcutaneous injection daily for 11 days (prevention study). In a reversal study, ACTH/saline was administered for 13 days and from day 8, apocynin or allopurinol was added for 5 days. Systolic blood pressure (SBP) was measured by the tail-cuff method and oxidative stress using plasma F2-isoprostane concentrations. Results were expressed as mean+/-SEM. RESULTS: ACTH increased SBP (P<.001) but apocynin or allopurinol alone had no effect. Apocynin (but not allopurinol) co-treatment prevented (142+/-3 ACTH, 120+/-4 mm Hg apocynin+ACTH, P'<0.001) and reversed ACTH-induced hypertension (133+/-4 to 118+/-5 mm Hg, P<.05). Plasma F2-isoprostane concentrations were increased in ACTH-treated rats compared with saline (11.9+/-1.0 vs 8.2+/-0.8 nmol/L, P<.01), and apocynin attenuated the ACTH-induced rise in plasma F2-isoprostane concentrations. Serum urate concentrations were undetectable in 75% of rats treated with allopurinol and were not affected by ACTH. CONCLUSIONS: Apocynin but not allopurinol prevented and reversed ACTH-induced hypertension in the rat. These results suggest superoxide production through NAD(P)H oxidase plays a role in ACTH-induced hypertension.

Optimizing microarray in experimental hypertension.

BACKGROUND: Genetic noise between outbred animals can potentially be a major confounder in the use of microarray technology for gene expression profiling. The study of paired organs from the same animal offers an alternative approach (e.g., for studies of the kidney in experimental hypertension). The present study was undertaken to determine the level of genetic noise between outbred adult Sprague-Dawley (SD) rats, and to determine the effects of unilateral nephrectomy on changes in gene expression as a basis for the design of microarray studies in experimental hypertension. METHODS: Male SD rats (approximately 130 g) were acclimatized before measurement of tail-cuff systolic blood pressure (SBP) for 6 control days and 4 days of saline treatment. Left kidney nephrectomy was performed, and the tissue snap-frozen in liquid nitrogen for subsequent RNA extraction. Two weeks later, SBP was measured over 4 control and 8 saline treatment days, and the remaining right kidney removed and frozen. Total RNA purification, preparation of cRNA, hybridization, and scanning of the Rat U34A Affymetrix arrays were performed, and data analyzed using MAS5 software Affymetrix Suite (v5), Bioconductor, as well as statistical methods motivated by relevant simulations. RESULTS: Gene expression profiles in the left control kidney were extremely consistent across animals. The expression profiles of pairs of kidneys from the same animal were, however, more similar than those of kidneys from different animals. Nephrectomy had little effect on the gene expression profiles in the time frame examined. CONCLUSION: Despite the outbred nature of the rats used in this study, they are useful for gene expression profiling comparisons. The use of paired organs from an individual animal ensures even further genetic identity, allowing determination of genes modified by the treatment of interest.

Nitric oxide donation lowers blood pressure in adrenocorticotrophic hormone-induced hypertensive rats.

Adrenocorticotrophic hormone (ACTH) elevates systolic blood pressure (SBP) and lowers plasma reactive nitrogen intermediates in rats. We assessed the ability of NO donation from isosorbide dinitrate (ISDN) to prevent or reverse the hypertension caused by ACTH. In the prevention study, male Sprague Dawley rats were treated with ACTH (0.2 mg/kg/day) or saline control for 8 days, with either concurrent ISDN (100 mg/kg/day) via the drinking water or water alone. Animals receiving ISDN via the drinking water were provided with nitrate-free water for 8 hours every day. In the reversal study ISDN (100 mg/kg) or vehicle was given as a single oral dose on day 8. SBP was measured daily by the indirect tail-cuff method in conscious, restrained rats. ACTH caused a significant increase in SBP compared with saline (P < 0.0015). In the prevention study, chronic administration of ISDN (100 mg/kg/day) did not affect the SBP in either group. In the reversal study, ISDN significantly lowered SBP in ACTH-treated rats at 1 and 2.5 hours (132 +/- 3 mmHg (1 h) and 131 +/- 2 mmHg (2.5 h) versus 143 +/- 3 mmHg (0 h), P < 0.002), but not to control levels. It had no effect in control (saline treated) rats. In conclusion, the lowering of SBP by NO donation is consistent with the notion that ACTH-induced hypertension involves an impaired bioavailability or action of NO in vivo.

Hemodynamic effects of the nitric oxide donor DETA/NO in mice.

(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO) is a recently synthesized member of NO-releasing, polyamine zwitterions, the so-called NONOates, that spontaneously liberate NO in aqueous solutions. The aim of this study was to determine the hemodynamic effects of DETA/ NO in normotensive and hypertensive mice. Male Swiss Outbred mice were implanted with TA11PA-C20 blood pressure devices (Data Sciences International, USA). After recovery (7-10 days), blood pressure was monitored for 10 days while mice were receiving saline (0.1 ml/20 g/day, s.c.). Mice were then treated every four hours for 1 day with either DETA/NO 60 mg/kg i.p. or the inactive metabolite, diethylenetriamine 38 mg/kg (molar equivalent) i.p. After a 2 week wash-out period, mice were treated with adrenocorticotrophic hormone (ACTH: 500 microg/kg/day, s.c.) for 10 days and re-challenged with DETA/NO or diethylenetriamine. Results were expressed as mean +/- SEM. After 10 days of saline treatment, baseline systolic and diastolic blood pressure (BP) were similar for animals subsequently receiving DETA/NO or the amine (123 +/- 1/95 +/- 3 and 124 +/- 1/92 +/- 0.2 mmHg) respectively. DETA/NO induced a profound fall in BP [Systolic: 74 +/- 4 mmHg (-40 +/- 3%); Diastolic: 46 +/- 4 mmHg (-52 +/- 4%)] and an increase in heart rate [729 +/- 33 bpm (32 +/- 2%)] within the first 80 minutes. Diethylenetriamine had no effect. ACTH treatment increased BP in both groups (137 +/- 16/108 +/- 12 and 161 +/- 1/142 +/- 1 mmHg) respectively. DETA/ NO induced a profound fall in blood pressure [Systolic: 92 +/- 11 mmHg (-32 +/- 7%); Diastolic: 68 +/- 10 mmHg (-35 +/- 10%)] and an increase in heart rate [613 +/- 36 bpm (18 +/- 6%)] within the first 80 minutes. Again diethylenetriamine had no significant effect. There was no significant effect on body weight with any treatment. Thus DETA/NO has potent blood pressure lowering effects in both normotensive and hypertensive mice.

Adrenocorticotropic hormone, blood pressure, and serum erythropoietin concentrations in the rat.

BACKGROUND: This study was designed to investigate the effects of adrenocorticotropic hormone (ACTH) on systolic blood pressure (BP) and serum erythropoietin (EPO) concentrations in two strains of rats. We hypothesized that ACTH-induced hypertension in the rat is characterized by increased EPO production. METHODS: Male Sprague-Dawley (SD) and Wistar out-bred (Wistar) rats were treated with saline or ACTH (0.2 mg/kg/d). Systolic BP was measured using the tail-cuff method. Serum EPO concentrations were assayed using an ELISA kit for human EPO, which cross-reacts with but underestimates rat EPO. Thymus weight was used as a marker of glucocorticoid activity. RESULTS: In SD rats, ACTH increased systolic BP (from 109 +/- 4 to 142 +/- 5 mm Hg, P <.0005), significantly greater than in saline-treated rats (P <.01). Systolic BP in ACTH-treated Wistar increased from 120 +/- 3 to 133 +/- 4 mm Hg (P <.05), but was not significantly different from saline-treated Wistar rats. The ACTH-induced increase in systolic BP was greater in SD than in Wistar rats (P <.05). Serum EPO levels were 5.6 +/- 0.4 in SD and 5.9 +/- 0.3 IU/L in Wistar rats, and decreased to undetectable levels with ACTH treatment in 10 of 10 SD and 7 of 10 Wistar rats. The ACTH treatment increased hemoglobin and hematocrit, and decreased thymus weight in both strains. CONCLUSIONS: 1) ACTH decreased serum EPO concentrations in both strains; 2) EPO is inversely related to ACTH-induced hypertension in the rat; and 3) Wistar rats are relatively resistant to the BP raising effects of ACTH treatment but not to the ACTH-induced decrease in thymus weight. These data suggest that EPO is not causal in ACTH-induced hypertension.

The antioxidant tempol prevents and partially reverses dexamethasone-induced hypertension in the rat.

BACKGROUND: Many forms of hypertension are associated with increased oxidative stress. This study investigated the effects of Tempol, a superoxide scavenger, on prevention and reversal of hypertension induced by the synthetic glucocorticoid dexamethasone (Dex) in the rat. METHODS: Male Sprague-Dawley rats (n = 10 in each group) were treated with saline or Dex (10 microg/kg/day subcutaneously) for 13 days. Tempol (1 mmol/L) was given in drinking water from 4 days before treatment (prevention) or from treatment day 8 (T8) (reversal). Systolic blood pressure (SBP) was measured by the tail-cuff method. Plasma F(2)-isoprostane concentrations were measured as a highly specific marker of oxidative stress. Thymus weight was measured as a marker of glucocorticoid activity. RESULTS: Dex treatment increased SBP (122 +/- 5 to 136 +/- 3 mm Hg, P <.05) and plasma F(2)-isoprostane concentrations (P =.005). Tempol alone did not alter SBP, but Tempol pretreatment prevented Dex-induced hypertension compared with that in rats treated with Dex alone (128 +/- 4 and 144 +/- 7 mm Hg respectively, P' <.05). Tempol partially reversed Dex-induced hypertension (122 +/- 5 and 136 +/- 3 mm Hg, respectively, P' =.057). Thymus weight was decreased in Dex-treated rats compared with saline treated rats (157 +/- 10 saline and 105 +/- 6 mg/100 g body weight Dex, P <.0005). Tempol affect neither thymus weight nor F(2)-isoprostane concentrations. CONCLUSIONS: Chronic Dex treatment increased SBP and tended to increase oxidative stress shown as increased plasma F(2)-isoprostane concentrations. Tempol prevented and partially reversed Dex-induced hypertension, independent of improvement in systemic oxidative stress measured by F(2)-isoprostane concentrations.

Lipopolysaccharide reverses adrenocorticotrophic hormone-induced hypertension in the rat.

Lipopolysaccharide (LPS) was used to stimulate nitric oxide (NO) release and investigate the effect of endogenous NO on adrenocorticotrophic hormone (ACTH)-induced hypertension in rats. After preliminary studies to determine the appropriate dose of LPS, 40 male Sprague-Dawley rats were treated with ACTH (200 microg/kg/day, s.c.) or saline (sham) for 8 days and then given a single dose of LPS (10 mg/kg, i.p.) or saline. ACTH treatment was continued for a further 5 days. Systolic blood pressure (SBP) was measured daily using the tail cuff method. Results were expressed as the mean +/- SEM. ACTH treatment significantly increased SBP (from 105 +/- 3 to 129 +/- 4 mmHg; p<0.05), whereas saline had no effect on SBP. The ACTH-induced increase in SBP was reversed by LPS injection (from 125 +/- 6 to 102 +/- 7 mmHg; p<0.05). SBP was also decreased in sham + LPS-treated rats compared with that of sham + saline-treated rats (p<0.05), but the SBP change in response to LPS was greater in ACTH-treated than in sham-treated rats (-23 vs. -8 mmHg; p<0.05). These data are compatible with the notion that reduced NO availability plays a role in ACTH-induced hypertension.

The anti-oxidant Tempol reverses and partially prevents adrenocorticotrophic hormone-induced hypertension in the rat.

OBJECTIVE: To investigate the effects of the antioxidant Tempol on prevention and reversal of adrenocorticotrophic hormone (ACTH)-induced hypertension in the rat, a model of hypertension characterized by nitric oxide deficiency. METHODS: Male Sprague-Dawley rats (n = 10 in each group) were treated with either saline or ACTH (0.2 mg/kg per day, s.c.) for 12 days. Tempol (1 mmol/l in drinking water) treatment was started on either day 8 (T8) of ACTH or saline treatment (reversal study), or 4 days prior to ACTH or saline treatment (prevention study). Systolic blood pressure (SBP) was measured using tail-cuff sphygmomanometry. Plasma F2-isoprostanes, a marker of oxidative stress, were measured by gas chromatography-mass spectrometry. RESULTS: ACTH increased SBP (mean +/- SEM: 119 +/- 5 to 147 +/- 7 mmHg, P < 0.0005) and plasma F2-isoprostane concentration (8.4 +/- 1.2 saline versus 12.9 +/- 1.6 nmol/l ACTH, P < 0.05). Tempol alone did not alter SBP, but administration of Tempol on T8 reversed ACTH-induced hypertension (from 134 +/- 4 T8 to 118 +/- 3 mmHg, P < 0.005). Tempol pre-treatment partially prevented ACTH-induced hypertension (123 +/- 2 mmHg, P' < 0.05). However, Tempol had no effect on F2-isoprostane concentrations at the dose used in this study. CONCLUSIONS: ACTH-induced hypertension in the rat is associated with increased oxidative stress. Tempol treatment reversed, and pretreatment partially prevented ACTH-induced hypertension, independent of improvement in systemic oxidative stress.

Decreased endothelial size and connexin expression in rat caudal arteries during hypertension.

OBJECTIVES: Hypertension is accompanied by endothelial dysfunction. The present study has investigated endothelial cell morphology and connexin expression in the caudal artery of the rat during the development of hypertension. METHODS: A significant increase in systolic blood pressure was detected from 9 weeks of age in spontaneously hypertensive male rats (SHR) compared to normotensive Wistar-Kyoto (WKY) rats, reaching a maximum by 11-12 weeks of age. Immunohistochemistry was used to quantify cell size and expression of connexins (Cxs) 37, 40 and 43 in the endothelium of prehypertensive (3-week-old) and hypertensive (12-week-old) rats. RESULTS: At 12 weeks, the size of endothelial cells and the expression of all three Cxs per endothelial cell were significantly less in SHR than WKY rats. At 3 weeks, there was no significant difference in cell size nor in the expression of Cxs 37 or 43; however, expression of Cx40 was significantly lower in SHR than in WKY rats. Between 3 and 12 weeks in WKY rats, there was no change in endothelial cell size, nor in the expression of Cxs 37, 40 and 43. In SHR, both cell size and Cx expression per endothelial cell were significantly decreased during the same developmental period, with a significant decrease in the density of Cx40 plaques. CONCLUSION: The development of hypertension in the SHR is accompanied by significant decreases in endothelial cell size and expression of Cx40, which may contribute to the endothelial dysfunction present in hypertension.