Angiotensin II is a crucial factor involved in regulation of both vascular tone and vascular structure (1,2). It has been demonstrated that the endothelium may modify the actions of angiotensin II. For example, vasoconstriction by angiotensin II in the renal circulation is counterbalanced by endogenous NO production, because inhibition of NO activity results in enhanced constrictor response to angiotensin II (3-5). This counterbalancing interaction between the vasoconstrictor angiotensin II and the vasodilator NO was also demonstrated in mesenteric resistance vasculature of both normotensive (6) and hypertensive (7) rats. More recently, angiotensin II was also shown to cause activation of endothelial nicotinamide adenine dinucleotide (NADH)-oxidase, resulting in endothelium-dependent superoxide production (8). This mode of action may inactivate NO at diffusion-limited rate (9). The potential clinical relevance of angiotensin II-mediated superoxide release has been underscored by the finding that the hypertensive action of angiotensin II can be partly reversed by coinfusion of (liposomal) superoxide dismutase (SOD), which rapidly degrades superoxide and consequently increases NO activity (10). No studies have been performed in humans to address these issues, nor is there information available on the relative contribution of these two opposing endothelial factors to the effects of angiotensin II in vivo. We therefore investigated to what extent the actions of angiotensin II on vasomotor tone are modulated by the endothelium in humans in vivo.
The first objective was to observe whether angiotensin II modulates endothelium-derived NO bioavailability in vivo. We therefore studied the effects of infusion of angiotensin II both during a "free" NO system and during a clamped NO system in the forearm. Clamping of the NO system was obtained by blocking endogenous NO with the selective blocker of NO synthase, NG-monomethyl-L-arginine (L-NMMA). Subsequently the L-NMMA-induced vasoconstriction was neutralized by infusion of an exogenous NO donor, sodium nitroprusside (SNP), until baseline forearm blood flow had been restored (11). If angiotensin II increases local NO production, then the vasoconstriction induced by angiotensin II is enhanced during the NO clamp. Second, to evaluate a potential contribution of angiotensin II-induced superoxide release, we evaluated the angiotensin II-induced vasoconstriction before and after coinfusion of vitamin C, a potent oxygen-radical scavenger (12). If angiotensin II increases local superoxide production, than vasoconstriction induced by angiotensin II is attenuated during vitamin C coinfusion.
We studied 26 healthy subjects (16 men), mean age, 23 years (standard deviation, 3 years). Subjects did not smoke and did not use medication. The study protocol was approved by the Utrecht University Hospital Ethics Committee for study in human beings. Subjects gave written informed consent after explanation of the protocol. Twelve hours before the forearm studies, all subjects refrained from smoking and drinking alcohol- or caffeine-containing beverages. Experiments were performed in a quiet room kept at a constant temperature of 22-24°C. The subjects were supine with both forearms resting slightly above heart level. A 22-g needle was inserted into the brachial artery of the nondominant arm after local anesthesia. Forearm blood flow (FBF) was measured in both arms at 10-s intervals by venous occlusion plethysmography with mercury-in-Silastic strain gauges with a microcomputer-based, R-wave-triggered system for online, semicontinuous monitoring (13). Upper-arm cuffs were inflated to 40 mm Hg. During FBF measurement, the hands were excluded from the circulation by wrist cuffs, inflated 40 mm Hg above systolic pressure. Intraarterial blood pressure (BP) was continuously monitored. Baseline measurements started ≥45 min after cannulation of the brachial artery, when FBF had stabilized. Between infusions, wrist cuffs were deflated, allowing ≥20 min for FBF to recover before each subsequent infusion. Infusions were given into the brachial artery: SNP (Merck, Darmstadt, Germany), L-NMMA (Institut fur Pharmazie, Universitat Leipzig, Germany), angiotensin II (Ang II; synthetic human, Cambridge Research Biochemicals Ltd, Cambridge, England), and vitamin C (Pharmachemie BV, Haarlem, Holland). The drugs were dissolved in 0.9% saline. All solutions were prepared aseptically from sterile stock solutions or ampules on the day of the study and stored at 4°C until use.
Angiotensin was administered in a cumulative dose of 0.014, 0.14, 0.35, and 1.4 ng/kg/min. Steady-state vasoconstriction was reached after 1.5 min, so each angiotensin dose was infused for 3 min. Calculations were based on measurements made during the final 1.5 min of each infusion step. In 12 subjects, this infusion block was repeated during coinfusion of vitamin C in a dose of 24 mg/kg/min, which has been reported effectively to scavenge oxyradicals (12). In 14 subjects, the infusion block was repeated during clamping of the NO system (11). Clamping was obtained by blocking endogenous NO production by L-NMMA infusion at a rate of 30 mg/kg/min. Subsequently, SNP was coinfused in incremental doses varying from 1 to 8 ng/kg/min, until baseline FBF had been restored (Fig. 1). We previously demonstrated that simultaneous infusion of L-NMMA and SNP results in a stable and sustained baseline FBF, which varies by <5% during 120 min of clamping (14). In five subjects, angiotensin II infusion was repeated during saline coinfusion as time control to exclude potential desensitization to angiotensin II on repeated infusion.
FBF was expressed as ml/100 ml forearm tissue/min. During each infusion step, the final six values of FBF from both measurement and control arm were used to calculate the mean FBF, and the ratio of FBF between measurement and control arm (M/C ratio; 15). Changes in M/C ratio are expressed as percentage decrease compared with baseline (Figs. 2 and 3). Results are expressed as mean ± SEM. Statistical analysis was performed by using two-way analysis of variance for repeated measures. A value of p < 0.05 was considered a significant difference.
Effect of NO clamp on angiotensin II-induced vasoconstriction
In 14 subjects, angiotensin II was infused during saline and during NO clamp. Cumulative dose infusion of angiotensin II caused an incremental vasoconstriction (FBF during saline, 2.70 ± 0.30 ml/100 ml forearm/min; dose I, 2.51 ± 0.31; dose II, 2.03 ± 0.22; dose III, 1.60 ± 0.18; and dose IV, 1.38 ± 0.15 ml/100 ml forearm/min). In the control arm, blood flow was not significantly altered. The ratios of the FBF in the measurement over control arm (M/C ratio) arm are shown in Fig. 2.
On L-NMMA infusion (30 mg/kg/min), FBF decreased by 31 ± 3%. Baseline forearm blood flow was restored by coinfusion of SNP at a mean dose of 2.4 ± 0.4 ng/kg/min (M/C ratio during saline, 1.04 ± 0.07, and during NO clamp, 1.05 ± 0.07). During NO clamp, vasoconstriction on cumulative-dose infusion of angiotensin II was significantly enhanced compared with saline coinfusion (FBF after restoration of baseline, 2.56 ± 0.25 ml/100 ml forearm/min; dose I, 2.16 ± 0.25; dose II, 1.66 ± 0.17; dose III, 1.28 ± 0.17; and dose IV, 1.19 ± 0.13 ml/100 ml forearm/min; p < 0.05, NO clamp vs. saline).
Effect of vitamin C on angiotensin II-induced vasoconstriction
In 12 subjects, angiotensin II was infused during saline and during vitamin C. Vitamin C alone did not significantly alter baseline FBF (FBF during saline, 2.57 ± 0.15 ml/100 ml forearm/min, and during vitamin C, 2.85 ± 0.24 ml/100 ml forearm/min; NS). During vitamin C, vasoconstriction on cumulative-dose infusion of angiotensin II was significantly attenuated compared with saline coinfusion (FBF during saline, 3.08 ± 0.33 ml/100 ml forearm/min; dose I, 2.70 ± 0.32; dose II, 2.12 ± 0.20; dose III, 1.55 ± 0.13; and dose IV, 1.12 ± 0.09 ml/100 ml forearm/min; FBF during vitamin C, 3.01 ± 0.23 ml/100 ml forearm/min; dose I, 2.71 ± 0.22; dose II, 2.30 ± 0.17; dose III, 1.81 ± 0.12; and dose IV, 1.61 ± 0.13 ml/100 ml forearm/min; p < 0.05 vitamin C vs. saline). M/C ratio curves are shown in Fig. 3.
In five subjects, angiotensin II infusion was repeated during coinfusion of saline (FBF during saline, 2.64 ± 0.22; dose I, 2.42 ± 0.24; dose II, 2.06 ± 0.16; dose III, 2.02 ± 0.24; dose IV, 1.60 ± 0.14; repeated administration: saline, 2.51 ± 0.27; dose I, 2.41 ± 0.23; dose II, 2.21 ± 0.20; dose III, 1.80 ± 0.18; dose IV, 1.55 ± 0.23; not significantly different first vs. second administration). Because no difference was observed, desensitization plays no role in the observed changes during vitamin C and clamping.
In this study, we demonstrate, for the first time in vivo in humans, that the actions of angiotensin II on vascular tone are acutely modulated by NO and reactive oxygen species. Thus in vivo angiotensin II causes a (counterregulatory) increase in NO release, as exemplified by a stronger angiotensin II-induced vasoconstriction during NO clamp. At the same time, coinfusion of vitamin C has an attenuating effect on angiotensin II-mediated vasoconstriction, implying angiotensin II-induced stimulation of superoxide production in human resistance vessels.
The vasoconstrictor response on infusion of angiotensin II is significantly increased after clamping of NO compared with free NO modulation, implying NO-mediated attenuation of angiotensin II-mediated vasoconstriction. In animal studies, a counterbalancing interaction between NO and angiotensin II was observed in the renal circulation (3-5,16), in the mesenteric vascular bed (6,7), and in the systemic circulation (17,18). In analogy, NO also was shown to counterbalance the action of other vasoconstrictive hormones, such as endothelin 1 and norepinephrine (19). These findings are in line with the concept that NO has a physiologic role as attenuating principle against vasoconstriction in general (20). In this respect, lack of further stimulation of an already increased NO production has been proposed as a failing counterregulatory mechanism in heart failure, leading to an inappropriate vasoconstriction in the presence of increased neurohumoral activation (21).
The NO-mediated inhibition of vasoconstriction during angiotensin II infusion may have several explanations. First, vasoconstriction induced by angiotensin II causes an increase in vessel wall shear stress. Shear stress has been shown to be a major stimulus for NO release in vivo (22,23). Second, angiotensin II also may increase NO production through shear stress-independent pathways. Angiotensin II-associated increase in NO bioavailability in isolated rings of rat carotid artery was abolished by both losartan, an AT1-receptor blocker, as well as nitro-L-arginine, a specific inhibitor of NO synthase (24). Because these experiments are performed under static conditions, shear stress can be excluded as stimulatory mechanism in this setup. The inhibition by losartan implies that the G protein-coupled AT1-receptor (25,26) can also be involved in mediating NO activation. Third, angiotensin II also was shown to stimulate both endothelial NO synthase messenger RNA (mRNA) expression (27,28) and activity of endothelial NO synthase (28) in rat renal tissue (27) and bovine pulmonary artery endothelium (28), respectively. However, in our study, the latter mechanism is probably not relevant. Finally, increased vasoconstriction by angiotensin II can be a consequence of decreased vascular sensitivity for exogenously infused nitroprusside during the clamp, because SNP was titrated before the infusion of angiotensin II was started. Mechanistically, this can only be related to increased oxygen radical production by angiotensin II (10). However, this final mechanism seems unlikely, because vascular responses to SNP in radical stress models in general remain unaltered (12,13).
Due to its rapid reaction with NO, superoxide has emerged as an important mediator of impaired NO-mediated vasodilatation (i.e., endothelial dysfunction). Vitamin C supplementation, either orally, intravenously, or local intraarterially, has been shown to ameliorate endothelial dysfunction in smokers, in diabetic patients, and in hypercholesterolemic patients by scavenging reactive oxygen species (12,29-31). In our study, vitamin C attenuated angiotensin II-induced vasoconstriction at higher dosages, whereas vitamin C had no effect on basal vascular tone. This would suggest that angiotensin II administration induces oxyradical release, whereas such oxyradicals contribute to the vasoconstrictor response. Indeed, Laurson (10) demonstrated in rats that angiotensin II-induced hypertension was in part mediated by increased vascular superoxide production. The increase in superoxide release could be prevented by pretreatment with losartan, demonstrating a role for the AT1 receptor in this processs (10). Losartan treatment in apo-E-deficient mice also attenuated low-density lipoprotein (LDL) cholesterol oxidation and thus atherosclerosis progression (32). Accordingly, angiotensin II results in significant upregulation of heme oxygenase I expression in rat aortic tissue, a known marker of the cellular redox state (33,34).
A limitation of infusion studies such as this one is that it is unknown to what extent infusion of angiotensin II reflects local tissue levels of angiotensin II. However, the calculated intraarterial concentrations (35) in our study varied from 3.7 × 10−12 to 3.7 × 10−9M, whereas local concentrations in heart, kidney, and vasculature have been reported in the 10−12-10−9M range (36,37). This would suggest that our observations indeed bear relevance for (patho)physiology.
It remains to be established whether the acute modulatory effects of NO and oxygen radicals on angiotensin II stimulation are beneficial or deleterious to vascular integrity. On the one hand, increased NO production may be protective against the vasoconstrictive and proliferative actions of angiotensin II and oxygen radicals. In this model, NO release is an integrated part of an endothelial "baroreceptor" system, which serves to control vascular tone. On the other hand, simultaneous stimulation of both NO production and reactive oxygen species by angiotensin II also may result in the production of peroxynitrite (38). Peroxynitrite is a potent oxidative substance that causes protein nitrosylation and fragmentation (39) and has been causally linked to the induction of endothelial dysfunction (38). In this respect, the current actions of angiotensin-II on NO and oxygen radicals would further reinforce its role as a pathogenetic factor in vascular disease. It also remains to be investigated whether the effects of angiotensin II on NO and oxygen radicals are specific or whether other vascular constrictor systems such as endothelins or catecholamines elicit similar responses.
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