Carrie A. Northcott

Endothelin-1 increases vascular superoxide via endothelin (A)-NADPH oxidase pathway in low-renin hypertension

Background—Angiotensin II–induced hypertension is associated with NAD(P)H oxidase–dependent superoxide production in the vessel wall. Vascular superoxide level is also increased in deoxycorticosterone acetate (DOCA)–salt hypertension, which is associated with a markedly depressed plasma renin activity because of sodium retention. However, the mechanisms underlying superoxide production in low-renin hypertension are undefined. Methods and Results—This study investigated (1) whether and how endothelin-1 (ET-1), which is increased in DOCA-salt hypertensive rats, contributes to arterial superoxide generation and (2) the effect of gene transfer of manganese superoxide dismutase and endothelial nitric oxide synthase. Both superoxide and ET-1 levels were significantly elevated in carotid arteries of DOCA-salt rats compared with that of the sham-operated controls. ET-1 concentration-dependently stimulated superoxide production in vitro in carotid arteries of normotensive rats. The increase in arterial superoxide in both ET-1–treated normotensive and DOCA-salt rats was reversed by a selective ETA receptor antagonist, ABT-627, the flavoprotein inhibitor diphenyleneiodonium, and the NADPH oxidase inhibitor apocynin but not by the nitric oxide synthase inhibitor N&ohgr;-l-arginine methyl ester or the xanthine oxidase inhibitor allopurinol. Furthermore, in vivo blockade of ETA receptors significantly reduced arterial superoxide levels, with a concomitant decrease of systolic blood pressure in DOCA-salt rats. Ex vivo gene transfer of manganese superoxide dismutase or endothelial nitric oxide synthase also suppressed superoxide levels in carotid arteries of DOCA-salt rats. Conclusions—These findings suggest that ET-1 augments vascular superoxide production at least in part via an ETA/NADPH oxidase pathway in low-renin mineralocorticoid hypertension.

Phosphoinositide 3-Kinase Mediates Enhanced Spontaneous and Agonist-Induced Contraction in Aorta of Deoxycorticosterone Acetate-Salt Hypertensive Rats

Abstract— Arteries from deoxycorticosterone acetate (DOCA)-salt and N&ohgr;-nitro-l-arginine (L-NNA) hypertensive but not normotensive rats develop spontaneous tone. LY294002 and wortmannin, phosphoinositide 3-kinase (PI3-kinase) inhibitors, eliminate spontaneous tone. We hypothesized that PI3-kinase protein and/or activity was increased in hypertension and contributed to the observed enhanced contractility. PI3-kinase activity assays revealed 2-fold higher activity in thoracic aorta from DOCA-salt [systolic blood pressure (SBP)=184±5 mm Hg] compared with sham rats (SBP=111±2 mm Hg). Western analyses of aortic homogenates revealed the presence of p85&agr;, p110&agr;, p110&bgr;, and p110&dgr; but not p110&ggr; PI3-kinase subunits; p110&dgr; protein was elevated in aorta of hypertensive rats as compared with sham. Aortic homogenates from L-NNA rats also had elevated p110&bgr; protein density, but neither L-NNA nor DOCA-salt had differences in p85&agr; and p110&agr;. Total Akt density was unaltered, but pAkt was significantly lower in homogenates from DOCA-salt rats. LY294002 (20 &mgr;mol/L) and nifedipine (50 nmol/L) abolished Ca2+-induced spontaneous tone in aorta from DOCA-salt rats. However, LY294002 did not alter BayK8644-induced contraction, indicating that LY294002 does not inhibit L-type Ca2+ channels directly. PTEN (phosphatase and tensin homolog) and pPTEN were expressed but not different in aorta from DOCA-salt and sham rats. LY294002 corrected the enhanced contraction to KCl and norepinephrine in aorta from DOCA-salt rats. These data support an increase in PI3-kinase activity and p110&dgr; density in aorta from L-NNA and DOCA-salt rats. Importantly, this increase contributes to the enhanced contractility observed in two models of hypertension.

The serotonin transporter is present and functional in peripheral arterial smooth muscle

We tested the hypothesis that the 5-HT transporter (5-HTT) is present and functional in peripheral arterial smooth muscle. In aorta and mesenteric resistance arteries, real time RT-PCR and western analyses indicated the presence of 5-HTT mRNA and a 74 kDa 5-HTT protein. Immunohistochemistry localized the transporter to smooth muscle and endothelial cells. 5-HT and the metabolite 5-hydroxyindole acetic acid (5-HIAA) were detected in aorta, carotid, and superior mesenteric arteries using HPLC; the MAOA inhibitor pargyline significantly increased (over 400%) arterial 5-HT concentration. 5-HT was taken up by arteries in a time-dependent manner and uptake was independent of the endothelium, sympathetic nerves, and norepinephrine transporter. 5-HT-induced contraction of normal aorta was potentiated by the 5-HTT inhibitor fluvoxamine. A change in arterial 5-HTT function occurs in deoxycorticosterone (DOCA)-salt hypertension as the potency and threshold of 5-HT in contracting aorta from the DOCA-salt rat was increased by fluoxetine and fluvox- amine (1 μmol/L; DOCA fluvoxamine −log EC50 [mol/L] = 6.85 ± 0.08, DOCA-control = 6.44 ± 0.08); expression of transporter was significantly increased in aorta of DOCA salt rats (145% Sham). These studies show for the first time the presence of the 5-HTT in peripheral arterial smooth muscle and raise the question as to the function of the 5-HTT in regulating peripheral effects of 5-HT.

PI3-Kinase Upregulation and Involvement in Spontaneous Tone in Arteries From DOCA-Salt Rats: Is p110δ the Culprit?

Abstract—Increased expression of phosphoinositide 3-kinase (PI3-kinase) mediates elevated tone in the aorta from hypertensive deoxycorticosterone acetate (DOCA)-salt rats. In this article, we hypothesized that (1) alterations observed with respect to PI3-kinase observed in the aorta would also occur in mesenteric resistance arteries responsible for determining total peripheral resistance (TPR) and (2) p110&dgr; activity was increased and localized to vascular smooth muscle cells (VSMCs), and was responsible for the increase in spontaneous tone in aortae from DOCA-salt rats. Mesenteric resistance arteries and aorta were isolated from DOCA-salt (190±3 mm Hg) and sham (121±2 mm Hg) rats. Myograph experiments revealed LY294002 (20 &mgr;mol/L), a PI3-kinase inhibitor, significantly decreased tone in mesenteric resistance arteries from DOCA-salt rats as compared with sham (−49±12 mg versus −10±7 mg). Western analyses of resistance artery protein homogenate revealed p85&agr; and p110&dgr; subunit protein, with significantly elevated levels of p110&dgr; protein in the DOCA-salt compared with sham rats (0.30±0.07 versus 0.16±0.04% smooth muscle alpha-actin arbitrary units). Immunohistochemistry revealed p110&dgr;-specific staining in VSMCs, with more intense staining in aortae from DOCA-salt rats. Compared with aortae from sham, p110&dgr;-associated PI3-kinase activity was increased in DOCA-salt (158% of sham) and likely responsible for spontaneous tone because the p110&dgr; specific inhibitor IC87114 decreased spontaneous tone in a concentration-dependent manner. Collectively, these data further implicate the p110&dgr; isoform of PI3-kinase in arterial hyperresponsiveness in hypertension at the level of both large and small arteries.

Endothelin‐1‐induced venous contraction is maintained in DOCA‐salt hypertension; studies with receptor agonists

Deoxycorticosterone acetate (DOCA) salt hypertension is associated with an endothelin‐1 (ET‐1)‐dependent increase in arterial resistance and mean circulatory filling pressure. Contraction of endothelium‐intact arteries and veins from sham and DOCA‐salt hypertensive rats to agonists of the ETA (ET‐1(1–31)) and ETB receptor (sarafotoxin 6c; S6c) was investigated in tissue baths as was expression of mRNA for ET‐1 and mRNA and protein for the ETA and ETB receptor. ET‐1(1–31) contracted aorta and vena cava from sham rats with a 30 fold lower potency than ET‐1. Contraction was not altered by the ETB receptor antagonist BQ788 (100 nM) but was abolished by the ETA receptor antagonist ABT‐627 (30 nM). In DOCA‐salt thoracic aorta, maximum contraction to ET‐1 and ET‐1(1–31) was reduced (36.6±6.3 and 13.3±4.4% of sham response, respectively); aorta did not contract to S6c. In vena cava from DOCA‐salt rats, contraction to ET‐1 and ET‐1(1–31) was not reduced compared to sham contraction; vena cava from sham and DOCA‐salt rats contracted to S6c with a similar potency. Real time RT–PCR revealed that prepro ET‐1 mRNA was increased 6.6±3.3 fold and 8.7±3.9 fold greater in DOCA‐salt aorta and vena cava, respectively, compared to sham. Vena cava expressed a higher content of ETA and ETB receptor mRNA than aorta (P<0.05), but no differences were observed between sham and DOCA‐salt tissues. ETA and ETB receptor protein was identified in all tissues. Immunoreactive ETA receptor, observed as a 65, 30 and 28 kDa bands, was expressed 400% greater in DOCA‐salt aorta compared to sham, but was not altered in vena cava. Immunoreactive ETB receptor, observed as 120, 45 and 30 kDa bands, tended to be higher in vena cava compared to aorta, but was not different in sham and DOCA‐salt vena cava. These results suggest that ETA receptor function is impaired in aorta but not vena cava of DOCA‐salt rats. The ETB receptor was present in the aorta but, unlike in veins, does not mediate contraction directly. A sustained response to ET‐1 in the venous circulation may contribute to the elevated blood pressure in the DOCA‐salt model.

Upregulated function of phosphatidylinositol-3-kinase in genetically hypertensive rats: a moderator of arterial hypercontractility.

1. The growth enzyme phosphatidylinositol 3‐kinase (PI3K) was recently implicated in the mediation of arterial spontaneous tone, an event observed in arteries from hypertensive, but not normotensive, subjects that contributes to changes in total peripheral resistance in the hypertensive state. We have shown this occurrence in experimentally induced models of hypertension. However, because the majority of hypertension is genetically based, it is important to demonstrate a similar change in genetically hypertensive animals.

ADENOVIRAL INHIBITION OF AT1A RECEPTORS IN THE PARAVENTRICULAR NUCLEUS INHIBITS ACUTE INCREASES IN MEAN ARTERIAL BLOOD PRESSURE IN THE RAT

Brain and peripheral renin-angiotensin systems are important in blood pressure maintenance. Circulating ANG II stimulates brain RAS to contribute to the increase mean arterial pressure (MAP). This mechanism has not been fully clarified, so it was hypothesized that reducing angiotensin type 1a (AT(1a)) receptors (AT(1a)Rs) in the paraventricular nucleus (PVN) would diminish intravenous ANG II-induced increases in MAP. Adenoviruses (Ad) encoding AT(1a) small hairpin RNA (shRNA) or Ad-LacZ (marker gene) were injected into the PVN [1 × 10(9) plaque-forming units/ml, bilateral (200 nl/site)] of male Sprague-Dawley rats instrumented with radiotelemetry transmitters for MAP and heart rate measurements and with venous catheters for drug administration. No differences in weight gain or basal MAP were observed. ANG II (30 ng·kg(-1)·min(-1) iv, 15 μl/min for 60 min) was administered 3, 7, 10, and 14 days after PVN Ad injection to increase blood pressure. ANG II-induced elevations in MAP were significantly reduced in PVN Ad-AT(1a) shRNA rats compared with Ad-LacZ rats (32 ± 6 vs. 8 ± 9 mmHg at 7 days, 35 ± 6 vs. 10 ± 6 mmHg at 10 days, and 32 ± 2 vs. 1 ± 5 mmHg at 14 days; P < 0.05). These observations were confirmed by acute administration of losartan (20 nmol/l, 100 nl/site) in the PVN prior to short-term infusion of ANG II; the ANG II-pressor response was attenuated by 69%. In contrast, PVN Ad-AT(1a) shRNA treatment did not influence phenylephrine-induced increases in blood pressure (30 μg·kg(-1)·min(-1) iv, 15 μl/min for 30 min). Importantly, PVN Ad-AT(1a) shRNA did not alter superior mesenteric arterial contractility to ANG II or norepinephrine; ACh-induced arterial relaxation was also unaltered. β-Galactosidase staining revealed PVN Ad transduction, and Western blot analyses revealed significant reductions of PVN AT(1) protein. In conclusion, PVN-localized AT(1)Rs are critical for short-term circulating ANG II-mediated elevations of blood pressure. A sustained suppression of AT(1a)R expression by single administration of shRNA can interfere with short-term actions of ANG II.

Low [Mg2+]e enhances arterial spontaneous tone via phosphatidylinositol 3-kinase in DOCA-salt hypertension

Abstract—Phosphatidylinositol 3-kinase (PI3K) has been implicated in low extracellular Mg2+ concentration ( [Mg2+]e)–induced aortic contraction, and Mg2+ deficiency has been associated with hypertension. Moreover, arterial PI3K activity is increased in hypertensive deoxycorticosterone (DOCA)-salt rats. We hypothesized that low [Mg2+]e activates PI3K, eliciting enhanced vascular contraction, PI3K activity, and norepinephrine (NE)-induced contraction. Spontaneous tone was monitored in endothelium-denuded aortic strips from sham and DOCA-salt rats exposed to low Mg2+ (0.15 mmol/L), high Mg2+ (4.8 mmol/L), or normal (1.17 mmol/L) physiologic salt solution (PSS) in isolated tissue baths. LY294002 (20 &mgr;mol/L), a PI3K inhibitor, or vehicle was added (30 minutes), followed by NE (10−9 to 3 x10−-5 mol/L). Low [Mg2+]e significantly enhanced tone in aortas from DOCA-salt and sham rats compared with normal PSS (DOCA-salt low [Mg2+]e, +51.5 +7.0 vs DOCA-salt normal PSS, +7.1 +1.4 % of initial phenylephrine [PE] contraction). LY294002 and incubation with high Mg2+ PSS decreased tone in aortas from DOCA-salt rats (low [Mg2+]e LY294002, −-87.5 +8.8; normal PSS LY294002, −81.7 +13.7; and high [Mg2+]e, −31.2 +10.8 % of initial PE contraction). Low [Mg2+]e leftward-shifted NE-induced aortic contractions in sham and thus matched the shift observed with DOCA (−log EC50 mol/L: sham PSS, −7.7 +0.1; DOCA-salt PSS, −8.2 +0.1; sham low [Mg2+]e, −8.2 +0.1; and DOCA-salt low [Mg2+]e, −8.1 +0.1). Moreover, this shift was inhibited by LY294002. In conclusion, low [Mg2+]e might activate PI3K, leading to enhanced tone and agonist-induced contraction observed in aortas from DOCA-salt hypertensive rats.

The Development of Hypertension and Hyperaldosteronism in a Rodent Model of Life-Long Obesity

Aldosterone has been linked to the deleterious cardiovascular effects of obesity in humans. The association of aldosterone with obesity in rodents is less well defined, particularly in models of diet-induced obesity. We hypothesized that adrenal aldosterone production and aldosterone synthase expression would be increased in rats with obesity-induced hypertension. Male Sprague Dawley rats were fed a high-fat (HF: 36% fat) or control diet from 3 wk of age, and mean arterial pressure (MAP) was measured by telemetry. MAP was increased after 4 wk of HF diet; this was 6 wk before changes in body weight. Mineralocorticoid receptor antagonism did not prevent the HF-induced increase in MAP. After 17 wk on the diets, HF rats had increased body and fat weights (abdominal and epididymal) and were insulin resistant (Homeostasis Model Assessment index: 3.53 ± 0.43 vs. 8.52 ± 1.77; control vs. HF, P < 0.05). Plasma aldosterone levels were increased in the HF rats (64.14 ± 14.96 vs. 206.25 ± 47.55 pg/ml; control vs. HF, P < 0.05). This occurred independently of plasma renin activity (4.8 ± 0.92 vs. 4.73 ± 0.66 ng/ml/h, control vs. HF). The increase in aldosterone was accompanied by a 2-fold increase in adrenal aldosterone synthase mRNA expression and zona glomerulosa hypertrophy. Rats were also studied after 8 wk of HF diet, a time when MAP, but not body weight, was increased. At this time plasma aldosterone was unchanged but plasma renin activity was increased (4.4 ± 0.5 vs. 8.1 ± 1.3 ng/ml/h; control vs. HF, P < 0.05). These studies suggest that rats fed a HF diet from weaning may be a useful model for studying obesity-associated hyperaldosteronism.

Chronic estradiol-17β exposure increases superoxide production in the rostral ventrolateral medulla and causes hypertension: Reversal by resveratrol

Women are exposed to estrogen in several forms, such as oral contraceptive pills and hormone replacement therapy. Although estrogen was believed to be cardioprotective, lately, its beneficial effects are being questioned. Recent studies indicate that oxidative stress in the rostral ventrolateral medulla (RVLM) may play a role in the development of hypertension. Therefore, we hypothesized that chronic exposure to low levels of estradiol-17β (E(2)) leads to hypertension in adult-cycling female Sprague Dawley (SD) rats potentially through generation of superoxide in the RVLM. To test this hypothesis, young adult (3 or 4 mo old) female SD rats were either sham-implanted or implanted (subcutaneously) with slow-release E(2) pellets (20 ng/day) for 90 days. A group of control and E(2)-treated animals were fed lab chow or chow containing resveratrol (0.84 g/kg of chow), an antioxidant. Rats were implanted with telemeters to continuously monitor blood pressure (BP) and heart rate (HR). At the end of treatment, the RVLM was isolated for measurements of superoxide. E(2) treatment significantly increased mean arterial pressure (mmHg) and HR (beats/min) compared with sham rats (119.6 ± 0.8 vs. 105.1 ± 0.7 mmHg and 371.7 ± 1.5 vs. 354.4 ± 1.3 beats/min, respectively; P < 0.0001). Diastolic and systolic BP were significantly increased in E(2)-treated rats compared with control animals. Superoxide levels in the RVLM increased significantly in the E(2)-treated group (0.833 ± 0.11 nmol/min·mg) compared with control (0.532 ± 0.04 nmol/min·mg; P < 0.05). Treatment with resveratrol reversed the E(2)-induced increases in BP and superoxide levels in the RVLM. In conclusion, these findings support the hypothesis that chronic exposure to low levels of E(2) induces hypertension and increases superoxide levels in the RVLM and that this effect can be reversed by resveratrol treatment.