Katja U.S. McKenzie

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.

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.

Role of anti-donor antibody in the rejection of pig proislet xenografts in mice.

Abstract: CBA/H mice produced serum anti‐pig IgG1, IgG2a, and IgG2b following xenotransplantation of pig proislets beneath the kidney capsule; anti‐pig IgM was present as pre‐existing antibody in the serum of normal CBA/H mice and was also produced in response to pig proislet xenografts. Serum anti‐pig IgG3was not detected post‐xenotransplantation. Rejection of pig proislet xenografts and the production of anti‐pig IgG1, IgG2a, and IgG2b isotypes were CD4 T cell‐dependent. The capacity for adoptively transferred hyperimmune CBA/H mouse anti‐pig PBL serum to induce the rejection of intact pig proislet xenografts in CD4 T cell‐depleted mice was dose dependent and correlated with markedly elevated levels of serum anti‐pig IgG3. Levels of anti‐pig IgG1, IgG2a, IgG2b, and IgM comparable to control mice acutely rejecting pig proislet xenografts and achieved following adoptive transfer of hyperimmune serum did not correlate with induced xenograft rejection. These findings suggest that anti‐pig Ig isotypes produced during the conventional process of acute proislet xenograft rejection do not play a major role in mediating graft damage. The acute rejection of pig proislet xenografts in the absence of serum anti‐pig Ig in μMT‐/‐ hosts confirmed that anti‐pig antibody is not essential for proislet xenograft destruction.

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.

Role of Tetrahydrobiopterin in Adrenocorticotropic Hormone‐Induced Hypertension in the Rat

Adrenocorticotropic hormone (ACTH)‐induced hypertension in the rat is characterized by nitric oxide deficiency. Tetrahydrobiopterin (BH4) is an essential cofactor for the enzyme nitric oxide synthase and glucocorticoids have been reported to reduce cytokine‐induced BH4 production. Accordingly we hypothesized that ACTH‐induced hypertension would be reversed by BH4 supplementation. Male Sprague–Dawley rats (n = 33) were treated with BH4 in vehicle (10 mg/kg/day i.p.) or vehicle alone (5 mg/kg/day i.p. of ascorbic acid in 4 mM HCl) for 10 days. ACTH (0.2 mg/kg s.c.) or saline daily injection was started 2 days after BH4 or vehicle treatment and continued for 8 days. Systolic blood pressure (SBP) was measured on alternate days using the tail cuff method. Treatment with HCl, ascorbic acid or BH4 alone had no effect on SBP. In saline treated rats, neither BH4 nor its vehicle modified SBP. In ACTH treated rats, SBP was increased in both BH4 (from 128 ± 6 to 142 ± 4 mmHg, T0 to T10, P < 0.0005, one way ANOVA) and vehicle groups (from 127 ± 3 to 158 ± 7 mmHg, T0 to T10, P < 0.001, one way ANOVA). There was no significant difference in SBP between BH4 + ACTH treated and vehicle + ACTH treated rats. Thus, daily injection of BH4 (10 mg/kg i.p.) failed to prevent the development of ACTH‐induced hypertension in rat.

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 µg/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.

Antioxidant Vitamins and Adrenocorticotrophic Hormone-Induced Hypertension in Rats

This study examined whether the anti-oxidants ascorbic acid, α- or γ-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) (200mg/kg/day drinking) or α-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, α- or γ-tocopherol (20 mg/kg/day) was added. ACTH increased SBP compared to saline (p < 0.05). AA or α-tocopherol failed to prevent and α- or γ-tocopherol failed to reverse ACTH-induced hypertension. Thus, neither vitamin C (water soluble) nor E (lipid soluble) modified ACTH-induced hypertension in the rat.

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.