SYMPOSIUM / SYMPOSIUM

Vascular biology of angiotensin and the impact ofphysical activity

James W.E. Rush and Crystal D. Aultman

Abstract: The renin–angiotensin system (RAS) is important for regulating blood pressure and extracellular fluid. The con-cept of the RAS has recently evolved from a classical systemic endocrine system to an appreciation of local RASs func-tioning in a paracrine manner, including in the vascular wall. Angiotensin II (AII), the main effector of the RAS, is apotent vasoconstrictor formed by the action of angiotensin-converting enzyme (ACE). ACE is multifunctional and also de-stroys the endogenous vasodilator bradykinin. A recently discovered novel ACE2 enzyme is responsible for forming a vas-odilatory compound, angiotensin 1–7, from AII. Thus, the actions of ACE and ACE2 are antagonistic. Tissue actions ofAII are mediated by specific receptors, AT1 and AT2, with AT1 mediating the classical actions. AT1-stimulated vasocon-stricton occurs via phospholipase-D-mediated second messenger generation directly, and indirectly via the coupling of AT1

to the prooxidant enzyme NADPH oxidase. Since the vascular NADPH oxidase is a major source of vascular reactive oxy-gen species generation and is responsible for the breakdown of the vasodilator nitric oxide (NO), there is another potentiallink between RAS and regulation of vasodilatory pathways. AT2 signaling is antagonistic to AT1 signaling, and results inbradykinin and NO formation. Chronic AII signaling induces vascular dysfunction, whereas pharmacological managementof the RAS can not only control blood pressure, but also correct endothelial dysfunction in hypertensives. Exercise trainingcan also improve endothelial function in hypertensives, raising the question of whether there is a potential role for RAS inmediating the vascular effects of exercise training. Recent studies have demonstrated reductions in the expression ofNADPH oxidase components in the vascular wall in response to exercise training, thus tempering one of the main cellulareffectors of AII, and this is associated with reduced vascular ROS production and enhanced NO bioavailability. Impor-tantly, it has now been demonstrated in human arteries that exercise training also tempers vascular AT1 receptor expressionand AII-induced vasoconstriction, while enhancing endothelium-dependent dilation. The signals responsible for thesechronic adaptations are not clearly understood, and may include changes in RAS components prompted by acute exercise.ACE genotype may have an effect on physical activity levels and on the cardiovascular responses to exercise training, andthe II genotype (compared with ID and DD) is associated with the largest endothelium-dependent dilations in athletes com-pared with those in sedentary individuals. Thus, the tissue location of the RAS, the complement of ACE/ACE2, the recep-tor expression of AT1/AT2, and the ACE genotype are all variables that could impact the vascular responses to exercisetraining, but the responses of most of these variables to regular exercise training and the mechanisms responsible have notbeen systematically studied.

Key words: vasomotor function, endothelium, vascular smooth muscle, exercise, angiotensin-converting enzyme, oxidativestress, ACE genotype, vascular health, hypertension.

Resume : Le systeme renine-angiotensine (RAS) joue un role important dans la regulation de la pression sanguine et duvolume de liquide extracellulaire. Traditionnellement considere comme partie integrante du systeme endocrinien, leconcept du RAS a evolue de telle sorte qu’on lui reconnaıt maintenant une fonction paracrine a action locale comme onl’observe dans les parois des vaisseaux sanguins. L’angiotensine II (AII), l’effecteur propre du RAS constitue un puissantvasoconstricteur obtenu par l’action de l’enzyme de conversion de l’angiotensine (ACE). L’ACE a plusieurs fonctionsdont celle de detruire la bradykinine, une substance endogene vasodilatatrice. L’ACE2, une enzyme recemment identifiee,catalyse a partir de l’AII la formation de l’angiotensine 1–7 a action vasodilatatrice. Ainsi donc, les actions de l’ACE etde l’ACE2 sont antagonistes. Les actions tissulaires de l’AII sont mediees par des recepteurs specifiques, AT1 et AT2, lepremier exercant son action classique. La vasoconstriction issue des AT1 depend de l’action d’un deuxieme messager, laphospholipase D, en ce qui concerne la voie directe et du couplage de AT1 a la NADPH oxydase, une enzyme pro-oxy-dante, en ce qui concerne la voie indirecte. Comme la NADPH oxydase vasculaire est une source importante d’especes re-actives oxygenees dans les vaisseaux et, en outre, catalyse la degradation de l’oxyde nitrique (NO), une substancevasodilatatrice, cela constitue un lien potentiel entre le RAS et les signaux associes a la vasodilatation. Les signaux emis

Received 25 January 2007. Accepted 6 June 2007. Published on the NRC Research Press Web site at apnm.nrc.ca on 22 December 2007.

J.W.E. Rush1 and C.D. Aultman. Department of Kinesiology, University of Waterloo, Waterloo, ON N2L 3G1, Canada.

1Corresponding author (e-mail: jwerush@uwaterloo.ca).

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par les recepteurs AT2 sont opposes a ceux des recepteurs AT1 et concourent a la formation de bradykinine et de NO.L’action chronique de AII entraıne un mauvais fonctionnement des vaisseaux sanguins et l’intervention pharmacologiqueaupres du RAS contribue non seulement a la regulation de la pression sanguine, mais a corriger les fonctions endothelialeschez les hypertendus. L’entraınement physique peut aussi ameliorer les fonctions endotheliales chez les hypertendus, cequi veut dire que le RAS aurait peut-etre un role de mediation dans les adaptations des vaisseaux sanguins a l’entraı-nement physique. Des etudes recentes ont observe une diminution de l’expression des constituants de la NADPH oxydasedans les parois des vaisseaux sanguins en reponse a l’entraınement physique, d’ou l’attenuation d’un des effets principauxde l’AII dans la cellule et la diminution de la production de ROS dans les vaisseaux sanguins et une amelioration de labiodisponibilite de NO. Tout aussi important, il est maintenant bien etabli que l’entraınement physique attenue aussi l’ex-pression des recepteurs AT1 dans les vaisseaux sanguins et la vasoconstriction causee par AII, ce qui ameliore la capacitede vasodilatation de l’endothelium. Les signaux responsables de ces adaptations chroniques ne sont pas bien compris etsemblent incorporer des modifications des constituants de RAS suscitees par l’exercice physique. Le genotype de l’ACEpeut aussi avoir un effet sur les niveaux d’activite physique requis et sur les adaptations cardiovasculaires a l’entraınementphysique ; dans le genotype II (comparativement a ID et DD), on observe une plus grande vasodilatation d’origine endo-theliale chez les athletes que chez les sedentaires. En conclusion, la localisation de RAS, les actions combinees de l’ACEet de l’ACE2, l’expression des recepteurs AT1 et AT2 de meme que le genotype de l’ACE sont toutes des variables qui in-fluencent les adaptations vasculaires a l’entraınement physique, mais les adaptations de la plupart de ces variables a l’en-traınement physique et les mecanismes responsables de ces adaptations n’ont pas fait l’objet d’etudes systematiques.

Mots-cles : fonction vasomotrice, endothelium, muscle lisse des vaisseaux sanguins, exercice, enzyme de conversion del’angiotensine, ECA, stress oxydatif, genotype, sante vasculaire, hypertension.

[Traduit par la Redaction]

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Physiological biochemistry of the renin–angiotensin system

The renin–angiotensin system (RAS) plays an essentialrole in the regulation of blood pressure and water and elec-trolyte homeostasis (Guyton and Hall 2000). The enzyme re-nin is released by the granular juxtaglomerular cells of thekidney in response to reductions in blood pressure, decreasesin distal tubule sodium chloride transport, and increases insympathetic tone. Renin cleaves angiotensinogen, an inac-tive plasma protein synthesized in the liver, to form the de-capeptide angiotensin I. Circulating angiotensin I (AI), inturn, is cleaved to the octapeptide angiotensin II (AII) by an-giotensin-converting enzyme (ACE; a dipeptidyl carboxy-peptidase), found in plasma, abundantly in pulmonary andvascular endothelial cells, and in the heart, kidneys, andbrain (Brewster and Perazella 2004; Jackson 2006; Siragy1999) (Fig. 1). ACE exists at the cell surface as an ectoen-zyme. The soluble (plasma) form of ACE results from theaction of ACE secretase, which cleaves the active portionof the ACE protein from the membrane-bound portion (Hall2003). AII is the main effector of the RAS, elevating arterialpressure rapidly as a result of its powerful vasoconstrictoraction, and on a longer time scale by its action on the kid-ney to decrease the excretion of salt and water, thus increas-ing the extracellular fluid volume. Angiotensinase enzymesin the tissue and blood inactivate AII.

Although the RAS was classically considered a circulatingsystem triggered by renin release and having general sys-temic effects, it is now recognized that there are also localtissue RASs that function in a more paracrine manner(Muller and Luft 1998; Siragy 1999). Thus, there is the pos-sibility for separation of the local and systemic effects ofRAS activation; the presence of local RAS allows tissueAII concentrations to be much higher than circulating con-centrations, and may place the sites of generation of AII incloser proximity to the effector tissues expressing AII recep-

tors, i.e., the AII target organs (Siragy et al. 1995). LocalRAS further affects the pharmacological management ofthis system because ACE inhibitors are more effective oncirculatory ACE than on tissue ACE, as a result of the bar-riers to drug access to its target. Local tissue AII concentra-tions are thus affected much less than circulatory AIIconcentrations during ACE therapy (Siragy et al. 1995).

AII formation persists during ACE inhibitor drug use be-cause a significant proportion (*40%) of AII is formed bynon-ACE-dependent pathways, either from angiotensinogenby the enzymes cathepsin G, elastase, and tissue plasmino-gen activator, or from angiotensin I by enzymes, includingchymase (Fig. 1; Brewster and Perazella 2004; Hollenberget al. 1998; Siragy 1999). Additionally, the more limited ac-cess of ACE inhibitors to tissue ACE compared with circu-lating ACE contributes to persistent AII formation duringACE inhibitor therapy.

Another curiosity related to ACE inhibitor therapy resultsfrom ACE’s multifunctional enzyme activity. In addition toits catalytic role in the formation of AII from AI, ACE alsocleaves and inactivates the naturally occurring vasodilatorsbradykinin and kallidin (Fig. 1; Fleming et al. 2005; Yanget al. 1970). Thus, inhibition of ACE is expected to notonly reduce AII levels and AII-induced vasoconstriction,but also to potentiate bradykinin-induced vasodilation. Thelatter effect may contribute to the observed antihypertensiveeffects of ACE therapy.

Recent developments in ACE enzymologyRecently, an ACE-related carboxypeptidase, ACE2, has

been identified (Donoghue et al. 2000; Fleming et al. 2005;Tipnis et al. 2000; Vickers et al. 2002). ACE2 shares *40%identity with the catalytic site of ACE, is highly expressedin vascular endothelial cells of the heart and kidney, andcan be released from the cell surface in a manner analogousto the process for ACE (Donoghue et al. 2000; Tipnis et al.

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2000). Although the general catalytic function of ACE2 issimilar to that of ACE, the specific petidyl cleavages cata-lyzed by ACE2 are different from those of ACE, and includecleavage of the carboxyl terminal phenylalanine residuefrom AII to form the vasodilator peptide angiotensin-(1–7)(Vickers et al. 2002). ACE2 may thus functionally antago-nize the classic RAS by both removing AII and producingangiotensin-(1–7). Furthermore, ACE2 is not sensitive toACE inhibitor drugs (Tipnis et al. 2000), and thus the in-fluence of ACE2 would be synergistic to ACE inhibitortherapy. Indeed, potential cardioprotective effects of ACE2expression have been observed in heart development and

disease models (Crackower et al. 2002), but an integrativeappreciation of the vascular biology of ACE2 and its con-tribution to control of vasomotor function has not been ex-plored.

Vascular AII receptors and intracellularsignaling

The tissue actions of AII are mediated by two specificcell-surface AII receptor types: AT1 and AT2. Both receptortypes have seven transmembrane domains, and are G-proteincoupled, although some cellular actions elicited by AT acti-

Fig. 1. Schematic illustration of the renin–angiotensin system (RAS) including tissue sites, enzymes, and intermediates, as well as functionalimpacts on blood vessels. ACE, angiotensin-converting enzyme, AT1, angiotensin II recptor type 1. Bottom inset illustrates the coupling ofAT1 to NADPH oxidase in vascular cells and the potential relationship between AII signaling and the nitric-oxide-mediated, endothelium-dependent dilation that this establishes.

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vation can be G-protein independent (Inagami et al. 1999;Kaschina and Unger 2003; Siragy 1999). Most of the tradi-tionally recognized actions of AII are mediated by AT1 re-ceptors (Allen et al. 2000). In addition to the renal effectsof increasing water and sodium retention (Goodfriend2000), AT1 receptor activation evokes G-protein-dependentand -independent responses in the arterial vascular smoothmuscle (VSM) (Kimura et al. 2004) including vasoconstric-tion, activation of the pro-oxidant enzyme NAD(P)H oxi-dase (Lopez et al. 2003), and stimulation of vascular cellgrowth (Wang et al. 2001), proliferation (Wolf and Wenzel2004), and extracellular matrix formation (Otsuka et al.1998). Since this review is focused on vasomotor effects ofAII, only the cell-signaling mechanisms pertinent to thisfunction will be discussed here. Other recent reviews containa broader perspective on AT receptor signaling and regula-tion (Inagami et al. 1999; Kaschina and Unger 2003; Siragy1999).

The constrictory effects of vascular smooth muscle AT1receptor activation occur through the activation of phospho-lipase C (PLC), causing the hydrolysis of phosphotidylinosi-tol 4,5-bisphosphate (PIP2) to form inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 signalingcauses elevation in myoplasmic Ca2+, and DAG signalingcauses protein kinase C activation. Both signaling eventsprompt VSM contraction. Synergistically, AT1-mediated in-hibition of adenylate cyclase depresses cAMP signaling,thus tempering VSM relaxation responses normally medi-ated by this second messenger (Siragy 1999). Other impor-tant indirect vasoconstrictory effects of AT1 activationoccur as a result of AII-induced augmentation of norepi-nephrine (NE) release from sympathetic nerve terminals andenhancement of the VSM contraction response to NE (Baltet al. 2001), as well as AII-mediated expression of thepowerful vasoconstrictor endothelin-1 (Hahn et al. 1993).AT1 activation also stimulates NADPH oxidase activity(Griendling et al. 2000) resulting in an elevated level of vas-cular reactive oxygen species (ROS), which reduces NO bi-oavailability and NO-dependent dilation (Rush et al. 2005),thus indirectly contributing to the net vasoconstrictory effectof AT1 receptor activation. The direct and indirect vasocon-strictory effects of AT1 activation are illustrated in Fig. 1.

While the AT2 receptor is prevalent in fetal tissue, impli-cating a possible role in growth and development, it is alsoexpressed at much lower levels in adults (Goodfriend 2000;Siragy 1999; Allen et al. 2000). Although less is known re-garding the regulation and action of the AT2 receptor, it isbecoming apparent that AT1 and AT2 can have antagonisticeffects. For instance, with respect to vascular distributionand vasomotor effects, some research suggests that the ef-fects of AT2 receptor activation promotes vasodilation andantiproliferation (Siragy 1999; Siragy et al. 1999). There aremultiple intracellular signaling networks and functional out-comes coupled to AT2 receptors, and much of the detail hasnot been well characterized. Part of the reason for the poorcharacterization of the AT2 receptor results from the morelimited distribution and density of AT2 vs AT1 and the an-tagonistic interaction of these two receptors (reviewed inInagami et al. 1999; Kaschina and Unger 2003; Siragy1999). The vasodilatory response to AT2 activation hasbeen shown to depend on AT2- dependent bradykinin pro-

duction, which, in turn, results in BK2 receptor activation,eNOS activation, NO production, and NO-mediated dilationthrough the cGMP mechanism in VSM (reviewed inKaschina and Unger 2003). This mechanism of AT2 recep-tor-mediated events has been confirmed in cultured endothe-lial cells and in the aortas of stroke-prone spontaneouslyhypertensive rats (Gohlke et al. 1998). To specifically inves-tigate the AT2-receptor-mediated effects of AII, the AT1 re-ceptor must be blocked. The sartan drugs are angiotensinreceptor blockers (ARB) that specifically block the AT1 re-ceptors, whereas the experimental drug PD 123319 is a se-lective ARB for the AT2 receptor. These agents have beenuseful not only clinically, but also experimentally in distin-guishing mechanisms of action of AT1 and AT2. The AII-evoked elevation in cGMP was shown to be inhibited byPD-123319, but not by losartan, confirming that this is anAT2-mediated event (Siragy et al. 1996; Siragy and Carey1999). The importance of this AT2 effect is also supportedby gene manipulation studies in which AT2 is eitherknocked out or overexpressed. Knockout mice lacking AT2have greater vasoconstrictory responses to AII and elevatedblood pressure, whereas AT2 overexpression induces vasodi-lation and activation of the vascular kinin – NO–cGMP sys-tem (Hein et al. 1995; Siragy et al. 1999; Tsutsumi et al.1999). Furthermore, AT2 receptor antagonism induces hy-pertension (Siragy and Carey 1999). Together, these resultsprovide functional evidence that AT2 mediates an importantvasodilatory role in adult animals. This could possibly be aprotective measure in normal blood pressure regulation andvascular homeostasis, especially when the RAS is chroni-cally activated, such as during hypertension and chronicheart failure. A true balancing and antagonism between AT1and AT2 actions seems to occur in terms of the vascular–vasomotor effects of AII (Inagami et al. 1999; Kaschinaand Unger 2003; Siragy 1999). Thus, a further character-ization of AT2 distribution and function in the adultcardiovascular system is a necessary step toward a compre-hensive understanding of the RAS.

Interaction of AII and nitric oxide-dependentvasomotor activity: NADPH oxidase

ROS contribute to intracellular signaling maintenance ofvascular integrity (Touyz 2004). They are produced by multi-ple enzymes, but quantitatively, NAD(P)H oxidase appearsto be the most significant source in the vasculature (Kojdaand Harrison 1999; Rajagopalan et al. 1996). Recent stud-ies and reviews of endothelial dysfunction in cardiovascu-lar disease have cited a major role for increased NAD(P)Hoxidase activation as a cause for reduced NO bioavailabil-ity and accelerated endothelial dysfunction in hypertension(Rajagopalan et al. 1996; Kerr et al. 1999; Graham andRush 2004; Rush et al. 2005) and other CV disease states(Lassegue and Clempus 2003; Ohara et al. 1993; Kim etal. 2002; Nedeljkovic et al. 2003). Thus, a possible site ofconvergence linking endothelial dysfunction with AII-dependent signaling processes is the regulation of NADPHoxidase.

NAD(P)H oxidases comprise both membrane-bound andcytosolic subunits that vary between vascular cell types. As-suming functional assembly of subunits, activation of

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NADPH oxidase is induced by multiple mechanical andchemical factors, including AII (De Keulenaer et al. 1998a;Chen and Keaney 2004; Brandes and Kreuzer 2005; DeKeulenaer et al. 1998b; Griendling et al. 2000). AII-inducedROS generation from NADPH oxidase is a biphasic re-sponse (Seshiah et al. 2002; Laplante et al. 2005). The ini-tial increase is initiated by AT1-dependent activation ofPLC, with DAG-dependent activation of PKC causing phos-phorylation of the NADPH oxidase subunit p47phox, andsubsequent increases in O2.

–. The second rise in ROS pro-duction is PKC independent and involves activation of thetyrosine kinase Src by H2O2, a O2.

– metabolite. Src phos-phorylates both epidermal growth factor (EGF) and phos-pholipase D (PLD). While EGF stimulates phosphoinositol-3-kinase promoting Rac activation, PLD and phospholipaseA2 (PLA2) increase formation of arachidonic acid (AA) andits metabolic byproducts. Rac, AA, and leukotrienes can di-rectly stimulate NAD(P)H oxidase (Brandes and Kreuzer2005; Seshiah et al. 2002; Siragy 1999). It is notable thatthis complex pathway creates a positive feedback loop initi-ated by AII, in which O2.

– enhances PKC activation, whileH2O2 activates both Src and EGF, which in turn enhanceROS production (Seshiah et al. 2002). This not only contrib-utes to the biphasic ROS response to AII, but the amplifica-tion created by this feedback loop could also help to explainwhy small changes in AII can result in large changes in vas-omotor functional effects, including a dramatic rise in bloodpressure.

While few studies have examined endothelial function inresponse to AII infusion, some attempts have been madeto elucidate the ROS and blood pressure-dependent and-independent mechanisms responsible for altered vasomotorfunction in models manipulating RAS and NADPH oxi-dase. Interruption of the gp91phox–p47phox interactionabolishes O2.

– production and attenuates systolic bloodpressure in AII-infused mice (Rey et al. 2001), suggestinga critical role for NADPH oxidase in the AII-mediated vas-cular dysfunction and hypertensive effects. AII-inducedO2.

– production is also inhibited in cultured endothelialcells from p47phox knockout mice, and is restored in thismodel by transfection of p47phox cDNA (Li and Shah2003). It is thus clear that the effectiveness of AT1-receptorinhibition for essential hypertension may be attributed, inpart, to reduced NAD(P)H oxidase activation and assem-bly, leading to diminished ROS production and enhancedNO bioavailability.

Using vasoconstrictors with different mechanisms of ac-tion, Laursen et al. (1997) demonstrated a separation ofblood pressure effects from the endothelial dysfunction andROS production effects accompanying AII action. Thus,whereas NE and AII infusion for 5 days caused a similarincrease in blood pressure, vascular O2.

– production was in-creased 3-fold by AII, but was not affected by NE, and en-dothelium-dependent dilation was impaired only in the AIIgroup (Laursen et al. 1997). In vivo administration of lipo-some-encapsulated SOD normalized O2.

– production, parti-ally restored endothelial-dependent relaxation, and partiallyattenuated blood pressure in AII-infused rats, but had no ef-fect in the NE-infused animals (Laursen et al. 1997). Simi-larly, although acute NE infusion had no effect on brachialartery dilation to Ach in healthy young men, acute infusion

of AII attenuated the ACh-induced dilatory response, andthe AII effect was abolished by simultaneous infusion ofthe antioxidant vitamin C (Hirooka et al. 2003). Collec-tively, these findings suggest both a pressure-independentmechanism for endothelial dysfunction involving reducedNO bioavailability, and a ROS- and NO-independent com-ponent of AII-induced hypertension per se.

Pharmacological management of RAS andvascular function

Because AII is a potent stimulus for many pathways im-plicated in hypertension, it has been the target of pharmaco-logical interventions, and the efficacy of ACE inhibitors andAT1 receptor blockers in attenuating blood pressure and en-dothelial dysfunction is well documented (e.g., Azevedo etal. 2003; Brosnan et al. 2002; Jackson 2006; Rodrigo et al.1997; Romero and Reckelhoff 1999). Whereas the literatureindicates that individually both ACE inhibitors and AT1 re-ceptor blockers are effective in controlling hypertension, theconcomitant administration of both drug types elicits moresubstantial improvements (Raasch et al. 2004) because ofdistinct biochemical outcomes. As previously mentioned,significant AII formation persists during ACE inhibition,and since ACE promotes metabolism of bradykinin (Fig. 1),ACE inhibition also increases bradykinin bioavailability.This has two effects: first, if AT1 blockers are also present,the persistent AII can interact with AT2 receptors and elicita significant vasodilation; and second, bradykinin is a potentvasodilator through the BK2 receptor coupled to eNOS acti-vation in vascular endothelium. These responses may opposeAT1-mediated constriction, thus contributing to the effec-tiveness these drugs. In contrast to ACE inhibitors, AT1receptor blockers do not evoke the same beneficial increasein bradykinin production, but they inhibit AT1-receptor-mediated responses and enhance AT2 activation by AII.

Although plasma AII concentration is elevated in diseasestates, including renovascular hypertension (Simon andAbraham 1995) and chronic heart failure (Brink et al.1996), more than 50% of people with essential hypertensionhave plasma AII levels comparable to those of normotensivepeople (Romero and Reckelhoff 1999), and hypertensive ratmodels can also have similar plasma AII levels as their re-spective normotensive counterparts (Sim and Qui 2003).However, in a seemingly paradoxical manner, both ACE in-hibitors and AT1 receptor blockers reduce blood pressureand restore endothelial function in these essential hyperten-sive patients and animals (Yavuz et al. 2003; Rodrigo et al.1997; Romero and Reckelhoff 1999). The solution to thisapparent paradox may lie in appreciating that, althoughplasma AII levels are not elevated above normal valuesfound in normotensive individuals, they are also not loweredby the prevailing cardiovascular conditions (e.g., increasedmean arterial pressure should lower renin release, RAS acti-vation, and AII production). The observation that this does notoccur in the cases mentioned prevents the re-establishmentof cardiovascular homeostasis and probably accounts forthe paradoxical findings (Simon and Abraham 1995;Romero and Reckelhoff 2000). A potential mechanism pro-posed to explain how near-normal plasma levels of AIImaintain chronic elevations in blood pressure is AII-in-

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duced activation of NAD(P)H oxidase, as outlined above,and enhancement of its expression (Seshiah et al. 2002).Thus, because of AII’s various mechanisms of action, thehistory of its exposure may affect the vascular biologicalresponses on different time scales and with different appa-rent sensitivities.

Chronic effects of AII on vascular phenotypeChronic in vivo AII infusion also increases the expression

levels of NAD(P)H oxidase subunits in both mouse and rataorta (Cifuentes et al. 2000; Mollnau et al. 2002), and theseincreases are attenuated by chronic administration of theAT1 receptor blocker irbesartan (Brosnan et al. 2002). Con-sistent with reported impairments in endothelial-independentdilation, chronic treatment with AII also reduces solubleguanylate cyclase (sGC) expression in rats, implying re-duced VSM sensitivity to NO (Laursen et al. 1997; Mollnauet al. 2002). Chronic AII infusion also increases eNOSprotein expression and activity in a variety of tissues(Hennington et al. 1998; Moreno et al. 2002; Mollnau et al.2002). These changes in eNOS may increase NO bioavail-ability and resist remodeling of the microvasculature as a re-sult of the inhibitory effect of NO on VSM proliferation.The expression and activity of extracellular superoxidedismutase (ecSOD) are also increased in AII- but not NE-infused mice, implying a pressure-independent mechanismof regulation; in organoid culture of mice aortic segments,this appears to be mediated through p42/44 mitogen acti-vated protein kinase (MAPK) (Fukai et al. 1999). IncreasedecSOD may preserve NO in the extracellular space throughwhich it must diffuse. Concomitant administration of theAT1 receptor blocker losartan abolishes all AII-inducedchanges in ecSOD. Thus, there is growing evidence thatchronic AII changes result in physiological and pathophy-siological alterations in cellular processes controlling vaso-motor function.

Although it is clear that NO, ROS, and AII serve criticalroles in normal physiological states, the antagonistic interac-tion of NO with AII and ROS demands an intricate balanceto maintain cardiovascular homeostasis (Schulman et al.2005). When the balance is disrupted, vascular dysfunctionresults. Chronic exercise training is a known stimulus forvascular adaptations and has been shown to improve im-paired vasomotor function (Rush et al. 2005; Kojda andHambrecht 2005). However, little is known regarding the in-fluence of exercise training on interactions of the angioten-sin system and NO bioavailability in the control of CVhomeostasis.

Angiotensin system and vascularadaptations to chronic exercise training

Exercise training elicits improvements in endothelial-dependent dilation in healthy controls and in a variety ofCVD models, including normotensive (Delp et al. 1993)and hypertensive rats (Graham and Rush 2004), hypercho-lesterolemic pigs (Thompson et al. 2004), and humans withessential hypertension (Higashi et al. 1999), chronic heartfailure (Hambrecht et al. 2003), and coronary artery dis-ease (Walsh et al. 2003). Some studies have explained

these findings, at least in part, by demonstrating that exer-cise is associated with changes in expression and activityof pathways influencing NO bioavailability (Kojda andHambrecht 2005; Rush et al. 2000, 2005). Exercise trainingfor 10 weeks also reduced the sensitivity of abdominalaortic rings to KCl-induced contraction by an endothelial-independent mechanism (Delp et al. 1993), and toNE-induced contraction by an endothelial-dependent mech-anism involving a2-adrenergic receptors (Spier et al. 1999;Delp et al. 1993). These findings lend potential insight intoanother possible mechanism of exercise training-inducedenhancements in NO bioavailability, because NE bindingto endothelial a2-adrenergic receptors increases intracellularCa2+, which in turn activates eNOS and promotes NO for-mation (Vanhoutte and Miller 1989). However, there is rel-atively little information regarding the influence of exercisespecifically on the vascular RAS or its role in vasodilatoryor vasoconstrictory adaptations to exercise training.

In addition to exercise training-dependent increases in theexpression and activity of vascular eNOS and antioxidantenzymes that favor enhanced NO bioavailability (Kojda andHambrecht 2005; Rush et al. 2005), growing evidence sup-ports the possibility that exercise induces adaptations inNADPH oxidase (Adams et al. 2005; Graham and Rush2004; Kojda and Hambrecht 2005; Rush et al. 2003, 2005),which, because of its importance as an effector of the AIIsignaling system, could interface the RAS with the vascularfunctional adaptations to exercise training. Related observa-tions to date include reductions in p67phox in isolated por-cine AEC (Rush et al. 2003) and reductions in gp91phox inrat thoracic aorta (Graham and Rush 2004) after chronicaerobic exercise training of moderate intensity. DiminishedmRNA expression of gp91phox, Nox4, and p22phox, aswell as decreased protein expression of gp91phox in humanmammary arteries are also found following 4 weeks of train-ing, and these changes are accompanied by attenuatedNAD(P)H oxidase activity and ROS production (Adams etal. 2005). In the latter study, the reduction in NADPH oxi-dase components was accompanied by decreases in vascularwall NADPH oxidase activity and ROS production (Adamset al. 2005). Thus, as NADPH oxidase can be consideredone of the main effectors of AII in the vascular tissue, thiseffect is significant in terms of the potential role of theRAS in the exercise training response. Indeed, the dilationof mammary artery segments to acetylcholine was enhancedin the trained vs the untrained group, and conversely, train-ing blunted the contractile response to AII in vitro (Adamset al. 2005).

As a possible explanation for the reduced maximalAII-induced vasoconstriction in human mammary arteriesfrom coronary artery disease patients following training,Adams et al. (2005) reported decreased AT1 receptor andincreased AT2 receptor mRNA in the mammary arteries oftrained vs untrained individuals. Whether coupled toNADPH oxidase or to the PLA2 mechanism of action, re-duced AT1 receptor expression would favor a blunted vaso-constrictor response to AII, consistent with the resultspresented (Adams et al. 2005). The universality of the ob-served exercise effect on vascular AT receptor expressionwill have to be assessed in various vascular beds and vessel

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types to fully evaluate the potential role of this adaptationin the mechanism of improved vascular function and re-duced vascular oxidative stress following exercise training.

Vascular adaptation to exercise may depend in part on theACE genotype. One polymorphism of the ACE gene is char-acterized by the presence (insertion, I) or absence (deletion,D) of a 287 bp segment in intron 16. The D allele is associ-ated with elevated tissue and circulating ACE activity(Defoor et al. 2006; Tanriverdi et al. 2005; Tiret et al.1992; Winnicki et al. 2004). Studies of the relationship be-tween physical activity and the ACE ID genotype have dem-onstrated that the ID polymorphism may be a specificgenetic factor associated with physical activity levels infree-living individuals, with sedentary lifestyle more com-mon among DD individuals than in II genotype borderlineand mild hypertensive people (Winnicki et al. 2004). Fur-thermore, in a population of coronary artery disease patients,the II genotype was associated with greater increases inaerobic power as a result of physical training in cardiac re-habilitation than those that occurred in patients with the IDor DD genotypes (Defoor et al. 2006). In the context of vas-cular function and exercise, a comparison of athletes withsedentary individuals within each genotype demonstratedthat athletes with the II ACE genotype had the greatest in-creases in endothelium-dependent flow-mediated dilation ofthe brachial artery, athletes with the DD ACE genotype hadthe smallest enhancement of flow mediated dilation, and theID heterozygotes had an intermediate response (Tanriverdiet al. 2005). Whether this reflects a contributing role forACE or angiotensin to the exercise training response in thefunction of the vascular endothelium, or reflects an unknownmechanism indirectly affiliated with the ACE genotype isunknown. A systematic study of the influence of the ACEgenotype on the trainability of the endothelium and themechanisms responsible would make a significant contribu-tion to the understanding of the mechanisms by which theRAS influences cardiovascular adaptations to exercise.

A variety of physical and chemical signals associated withexercise may prompt the molecular phenotypic changes thatin turn control vascular functional adaptations to exercise(Adams et al. 2005; Kojda and Hambrecht 2005; Rush et al.2005). AII may be a potential candidate for a chemical me-diator of exercise adaptations, because AII infusion studieshave confirmed that some of the effects of AII exposure aresimilar to responses prompted by exercise training, such asenhanced vascular eNOS and ecSOD expression (Fukai etal. 1999; Graham and Rush 2004; Hennington et al. 1998;Kojda and Hambrecht 2005; Moreno et al. 2002).

Although studies of acute exercise effects on AII levelshave been performed on subjects with a variety of compli-cating conditions, in general, their results all demonstratethat there is an elevation of tissue and circulating AII in re-sponse to an exercise bout (Aldigier et al. 1993; Braith et al.1999; Kinugawa et al. 1997; Kosunen and Pakarinen 1976;Miura et al. 1994; Woods et al. 2004) which may or maynot (Aldigier et al. 1993; Miura et al. 1994) be ACEdependent. Renin activity (Kosunen and Pakarinen 1976;Milledge and Catley 1982) and AI levels (Arvay et al.1982) have likewise been demonstrated to increase as a re-sult of acute exercise. One particularly well controlled study

demonstrated that AII was elevated several-fold within10 min of beginning 70% VO2 max cycling exercise and itcontinued to rise over the next 10 min. Upon exercise cessa-tion, although AII dropped within the first 10 min, it re-mained elevated at levels several-fold above resting levelsfor at least 40 min of recovery (Woods et al. 2004). Thepersistent elevation of AII during and after acute exercise inthis study suggests that this molecule could influence vascu-lar molecular phenotype and function via known effects ofAII (Fukai et al. 1999; Hennington et al. 1998; Moreno etal. 2002). Acute exercise and exercise training studies usingARB and (or) ACE inhibitors would be useful in testing thishypothesis that AII dynamics during acute exercise boutscontribute to exercise training-induced vascular phenotypicadaptations. Whether these adaptations would occur via a di-rect effect of AII signaling on gene expression, or indirectlythrough AII-induced ROS, could be determined with antiox-idant manipulations or NADPH knockout animals under-going exercise protocols. However, because both basallevels and the degree of acute exercise-induced elevationsin AII were independent of the ACE ID genotype in severalstudies (Chadwick et al. 1997; Danser et al. 1999; Woods etal. 2004), it is not likely that the hypothesized role for AIIcould account for the genotype-dependent improvements inendothelium-dependent flow-mediated dilation in athletescompared with those in sedentary individuals described pre-viously (Tanriverdi et al. 2005).

Perspective

The RAS is a complex system with systemic and local en-docrine and paracrine effects. The complexity is not limitedto location, as there are multiple forms of carboxypeptidaseswith ACE-type function that result in the generation of a va-riety of substances with both vasoconstrictory and vasodila-tory effects. Likewise, the two main types of AII receptorsalso have antagonistic effects, so the expression pattern ofthese receptors in the tissue of interest, and any changes inthe expression of these receptors in response to physiologi-cal and pathophysiological stimuli, can change the tissue re-sponse to AII. Because of the intracellular targets of AT1receptor signaling, there is a relationship between AII sig-naling and NO bioavailability via NADPH oxidase. Prelimi-nary observations indicate improvement in endothelium-mediated dilation; tempering of AII-induced constriction,NADPH oxidase expression, NADPH activity, and NADPHROS production; and blunted expression of AT1 in arteriesafter training. The signals coordinating adaptations of vascu-lar phenotype and function to exercise training and the po-tential involvement of RAS components are not known,although it can be speculated that AII might contribute, asthis molecule responds to acute exercise bouts and is knownto cause gene expression changes in vascular cells that, insome cases, are similar to those resulting from exercisetraining. Furthermore, the ACE genotype may be a determi-nant of the vascular response to exercise training. The com-plexity of the RAS and its potential impact on vascularfunction justifies efforts to fully describe the vascular RASalterations with exercise training and determine their impor-tance to the establishment of the functional adaptations of

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arteries to exercise that underlie changes in vasomotor activ-ity, regulation of blood flow, and blood vessel structure.

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