Gregory D. Fink

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.

Area postrema is critical for angiotensin-induced hypertension in rats.

The effect of surgical ablation of the area postrema on acute (5-10 minutes) and chronic (5-10 days) increases in mean arterial pressure produced by intravenous infusion of angiotensin II in conscious, instrumented rats was studied. In agreement with previous studies, pressor responses of area postrema-ablated rats (n = 11) to acute angiotensin II infusion were identical to those of control sham-lesioned rats (n = 13). In these same rats, however, a 5-day infusion of angiotensin II produced a sustained hypertension in the sham-lesioned group whereas mean arterial pressure was increased only transiently (1-3 days) in the area postrema-ablated rats. No differences before infusion of arterial pressure, heart rate, water intake, urinary sodium excretion, and urinary potassium excretion were observed between sham-lesioned and area postrema-ablated rats; only arterial pressure was changed significantly during angiotensin II infusion in either group. Twenty-four hours after terminating angiotensin II infusion, mean arterial pressure was within the normotensive range in both sham-lesioned and area postrema-ablated rats. In a separate group of sham-lesioned (n = 13) and area postrema-ablated (n = 12) rats, angiotensin II was infused intravenously for a 10-day period; mean arterial pressure was increased significantly over the entire 10-day infusion in sham-lesioned rats, but for only 1 day in area postrema-ablated rats. An intact area postrema appears necessary for the development of chronic, but not acute, hypertension during intravenous infusion of angiotensin II in the rat.

Gene Transfer of Human Guanosine 5′-Triphosphate Cyclohydrolase I Restores Vascular Tetrahydrobiopterin Level and Endothelial Function in Low Renin Hypertension

Background—We recently reported that arterial superoxide (O2−) is augmented by increased endothelin-1 (ET-1) in deoxycorticosterone acetate (DOCA)-salt hypertension, a model of low renin hypertension. Tetrahydrobiopterin (BH4), a potent reducing molecule with antioxidant properties and an essential cofactor for endothelial nitric oxide synthase, protects against O2−–induced vascular dysfunction. However, the interaction between O2− and BH4 on endothelial function and the underlying mechanisms are unknown. Methods and Results—The present study tested the hypothesis that BH4 deficiency due to ET-1–induced O2− leads to impaired endothelium-dependent relaxation and that gene transfer of human guanosine 5′-triphosphate (GTP) cyclohydrolase I (GTPCH I), the first and rate-limiting enzyme for BH4 biosynthesis, reverses such deficiency and endothelial dysfunction in carotid arteries of DOCA-salt rats. There were significantly increased arterial O2− levels and decreased GTPCH I activity and BH4 levels in DOCA-salt compared with sham rats. Treatment of arteries of DOCA-salt rats with the selective ETA receptor antagonist ABT-627, NADPH oxidase inhibitor apocynin, or superoxide dismutase (SOD) mimetic tempol abolished O2− and restored BH4 levels. Basal arterial NO release and endothelium-dependent relaxations were impaired in DOCA-salt rats, conditions that were improved by apocynin or tempol treatment. Gene transfer of GTPCH I restored arterial GTPCH I activity and BH4 levels, resulting in reduced O2− and improved endothelium-dependent relaxation and basal NO release in DOCA-salt rats. Conclusions—These results indicate that a BH4 deficiency resulting from ET-1–induced O2− via an ETA/NADPH oxidase pathway leads to endothelial dysfunction, and gene transfer of GTPCH I reverses the BH4 deficiency and endothelial dysfunction by reducing O2− in low renin mineralocorticoid hypertension.

Splanchnic Circulation Is a Critical Neural Target in Angiotensin II Salt Hypertension in Rats

Chronic angiotensin II (Ang II) infusion, in rats fed high salt, engages the sympathetic nervous system to increase venomotor tone. The splanchnic sympathetic nervous system is the most important regulator of venous tone, indicating that splanchnic sympathetic nervous system activity may be increased in Ang II salt hypertension. We hypothesized that celiac ganglionectomy (CGx), to selectively disrupt sympathetic innervation to the splanchnic circulation, would attenuate arterial pressure (AP), and venous tone increases in Ang II salt hypertension. Rats fed 2% or 0.4% NaCl were instrumented to allow AP measurement by radiotelemetry at the same time as surgical CGx or sham operation. Ang II was delivered by minipump (150 ng/kg per minute) for 14 days. CGx reduced AP independent of salt diet during control. CGx markedly attenuated Ang II hypertension in rats on 2% NaCl but had little effect in rats fed 0.4% NaCl. To test the possibility that CGx exerted its effects via renal denervation, rats were subjected to the same protocol but received selective bilateral renal denervation. Renal denervation decreased AP during control but had no protective effect on Ang II hypertension and actually tended to exacerbate the pressor response. Finally, separate groups of rats underwent CGx or sham operation and were instrumented to allow repeated measures of mean circulatory filling pressure, an index of venous tone. In addition to attenuating Ang II salt hypertension, CGx completely prevented Ang II salt-induced increases in mean circulatory filling pressure and substantially attenuated depressor responses to acute ganglion blockade. We conclude that, in the presence of high salt, Ang II activates the splanchnic sympathetic nervous system to increase venomotor tone and AP.

LONG-TERM SYMPATHO-EXCITATORY EFFECT OF ANGIOTENSIN II : A MECHANISM OF SPONTANEOUS AND RENOVASCULAR HYPERTENSION

1. The peptide hormone angiotensin II (AngII) is acknowledged to be an important factor in the pathophysiology of hypertension. This is particularly the case in hypertension caused by luminal narrowing of one renal artery, (i.e. renovascular hypertension). The primary mechanism by which AngII raises blood pressure, however, is disputed. Strong arguments can be made supporting either vascular contraction, effects on renal excretion of sodium and water, or trophic actions on cardiovascular structures as the key element. In this paper I review evidence that AngII influences blood pressure by modulating autonomic nervous system activity. Modulation can occur at both the peripheral and central aspects of the autonomic system, but I focus on brain pathways involved in determining sympathetic nervous system activity.

Tempol Lowers Blood Pressure and Sympathetic Nerve Activity But Not Vascular O2− in DOCA-Salt Rats

Abstract—This study tested the hypothesis that depressor responses caused by tempol are not associated with reductions in vascular O2− levels in urethane-anesthetized deoxycorticosterone acetate (DOCA)-salt hypertensive rats. We compared the effects of intravenous (IV) administration of tempol, apocynin, superoxide dismutase-polyethylene glycol (PEG-SOD), and SOD on mean arterial blood pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA). In DOCA-salt rats, tempol (30 to 300 &mgr;mol/kg) dose-dependently decreased RSNA, MAP, and HR. Tempol (300 &mgr;mol/kg) decreased MAP from 140±5 to 83±4 mm Hg (P <0.05). HR decreased from 435±15 to 390±12 bpm (P <0.05). RSNA was reduced by 54%±6% from baseline. However, in the same rats, tempol did not reduce dihydroethidium-induced fluorescent signals in the aorta and vena cava. Apocynin (200 &mgr;mol/kg) did not lower MAP (142±5 mm Hg versus 140±6 mm Hg) or HR (428±15 bpm versus 420±13 bpm) and apocynin did not potentiate depressor responses caused by tempol. PEG-SOD (10 000 U/kg, bolus or 5000 U/kg bolus followed by a 30-minutes infusion of 500 U/kg/min) or SOD (25 000 U/kg, bolus or 10 000 U/kg bolus followed by a 30-minutes infusion of 1000 U/kg per minute) did not alter MAP or HR. It is concluded that depressor responses and decreases in HR and RSNA caused by acute tempol treatment are caused by direct sympathetic nerve activity inhibition that is not accompanied by SOD-mimetic action in the aorta or vena cava.

Mechanisms of increased venous smooth muscle tone in desoxycorticosterone acetate-salt hypertension

The purpose of the present study was to identify mechanisms that contribute to increased venous smooth muscle tone in desoxycorticosterone acetate (DOCA)-salt hypertension in rats. Male Sprague-Dawley rats were uninephrectomized, received subcutaneous implants of DOCA, and drank 1% sodium chloride/0.2% potassium chloride solutions. Sham-operated rats received only uninephrectomy and drank tap water. Three to 4 weeks later, arterial and venous catheters were implanted for measurements of arterial and central venous pressures, respectively, and a silicone balloon catheter was permanently fixed in the right atrium to produce brief circulatory arrest. Venous smooth muscle activity was estimated on the basis of repeated measurements of mean circulatory filling pressure in conscious rats resting in their home cages. DOCA-salt-treated rats were hypertensive and had elevated mean circulatory filling pressure compared with normotensive sham-operated rats. Blockade of the endothelin subtype A receptor with 1 mg/kg ABT-627 IV decreased arterial blood pressure and mean circulatory filling pressure significantly more in hypertensive rats than in normotensive rats. Ganglionic blockade with 30 mg/kg hexamethonium IV also decreased arterial blood pressure and mean circulatory filling pressure more in hypertensive than in normotensive rats. Pretreatment with ABT-627 did not affect subsequent hemodynamic responses to ganglionic blockade. We conclude that venous smooth muscle tone is increased in DOCA-salt hypertension through the independent actions of both endogenous endothelin-1 acting on subtype A receptors and sympathetically mediated venoconstrictor activity.

Chronic Angiotensin II Infusion Causes Differential Responses in Regional Sympathetic Nerve Activity in Rats

Angiotensin II (AngII)–induced hypertension in experimental animals has been proposed to be attributed in part to activation of the sympathetic nervous system. This sympathetic activation appears to be accentuated in animals consuming a high-salt diet (AngII-salt hypertension). However, accurate quantification of sympathetic activity is difficult, and controversy remains. It is particularly important to ask which are the critical vascular beds targeted by increased sympathetic nerve activity (SNA) in AngII-salt hypertension. To address this issue, mean arterial pressure and renal SNA or lumbar SNA were continuously recorded during a 5-day control period, 11 days of AngII (150 ng/kg per minute, SC), and a 5-day recovery period in conscious rats on a high-salt (2% NaCl) diet. Although mean arterial pressure reached a new steady-state level of 30 to 35 mm Hg above control levels by the end of the AngII period, renal SNA decreased by 40% during the first 7 days of AngII and then returned toward control levels by day 10 of AngII. In contrast, lumbar SNA remained at control levels throughout the AngII period. In another experiment we measured hindlimb norepinephrine spillover in conscious rats on normal (0.4%) or high- (2.0%) salt diets before and during 14 days of AngII administration. AngII had no significant affect on hindlimb norepinephrine spillover in either group. We conclude that chronic AngII modulates renal and lumbar SNAs differentially in rats consuming a high-salt diet and that AngII-salt hypertension in the rat is not caused by increased SNA to the renal or hindlimb vascular beds.

NADPH oxidase-derived superoxide augments endothelin-1-induced venoconstriction in mineralocorticoid hypertension

Abstract—Deoxycorticosterone acetate (DOCA)–salt hypertension is characterized by low renin/angiotensin but increased arterial superoxide levels. We have recently reported that the arterial endothelin-1 (ET-1) level is increased, resulting in NADPH oxidase activation and superoxide generation. However, the effect of ET-1 on venous superoxide production and its relation to venoconstriction are unknown. The present study tested the hypotheses that ET-1 stimulates venous NADPH oxidase and superoxide via its ETA receptors, resulting in enhanced venoconstriction in DOCA-salt hypertensive rats. Treatment with ET-1 (0.01 to 1 nmol/L), but not the selective ETB receptor agonist sarafotoxin s6c, of vena cavas of normal rats concentration-dependently increased superoxide levels, an effect that was abolished by the selective ETA receptor antagonist ABT-627. Although the ET-1 level was not increased in the vena cava and plasma, both venous NADPH oxidase activity and superoxide levels were significantly higher in DOCA-salt compared with sham rats. Moreover, ET-1 treatment (10−9 mol/L, 10 minutes) of isolated vena cavas further elevated superoxide levels in DOCA-salt rats only but not sham rats, an effect that was abrogated by the superoxide scavenger tempol. Similarly, ET-1–induced contractions of isolated vena cavas of DOCA-salt but not sham rats were significantly inhibited by tempol. The NADPH oxidase inhibitor apocynin significantly reduced superoxide levels in vena cavas of DOCA-salt rats and in ET-1–treated vena cavas of normal rats. Finally, in vivo ETA receptor blockade by ABT-627 significantly lowered venous superoxide levels and blood pressure in DOCA-salt but not sham rats. These results suggest that superoxide contributes to ET-1–induced venoconstriction through an elevated venous NADPH oxidase activity in mineralocorticoid hypertension.

Region-specific changes in sympathetic nerve activity in angiotensin II–salt hypertension in the rat

It is now well accepted that many forms of experimental hypertension and human essential hypertension are caused by increased activity of the sympathetic nervous system. However, the role of region‐specific changes in sympathetic nerve activity (SNA) in the pathogenesis of hypertension has been difficult to determine because methods for chronic measurement of SNA in conscious animals have not been available. We have recently combined indirect, and continuous and chronic direct, assessment of region‐specific SNA to characterize hypertension produced by administration of angiotensin II (Ang II) to rats consuming a high‐salt diet (Ang II–salt hypertension). Angiotensin II increases whole‐body noradrenaline (NA) spillover and depressor responses to ganglionic blockade in rats consuming a high‐salt diet, but not in rats on a normal‐salt diet. Despite this evidence for increased ‘whole‐body SNA’ in Ang II–salt hypertensive rats, renal SNA is decreased in this model and renal denervation does not attenuate the steady‐state level of arterial pressure. In addition, neither lumbar SNA, which largely targets skeletal muscle, nor hindlimb NA spillover is changed from control levels in Ang II–salt hypertensive rats. However, surgical denervation of the splanchnic vascular bed attenuates/abolishes the increase in arterial pressure and total peripheral resistance, as well as the decrease in vascular capacitance, observed in Ang II–salt hypertensive rats. We hypothesize that the ‘sympathetic signature’ of Ang II–salt hypertension is characterized by increased splanchnic SNA, no change in skeletal muscle SNA and decreased renal SNA, and this sympathetic signature creates unique haemodynamic changes capable of producing sustained hypertension.