SYMPOSIUM / SYMPOSIUM

Vascular biology of angiotensin and the impact of

physical activity
James W.E. Rush and Crystal D. Aultman
Abstract: The reninangiotensin 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 a
potent 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 17, from AII. Thus, the actions of ACE and ACE2 are antagonistic. Tissue actions of
AII 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 potential
link between RAS and regulation of vasodilatory pathways. AT
2
signaling is antagonistic to AT
1
signaling, and results in
bradykinin and NO formation. Chronic AII signaling induces vascular dysfunction, whereas pharmacological management
of the RAS can not only control blood pressure, but also correct endothelial dysfunction in hypertensives. Exercise training
can also improve endothelial function in hypertensives, raising the question of whether there is a potential role for RAS in
mediating the vascular effects of exercise training. Recent studies have demonstrated reductions in the expression of
NADPH oxidase components in the vascular wall in response to exercise training, thus tempering one of the main cellular
effectors 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 expression
and AII-induced vasoconstriction, while enhancing endothelium-dependent dilation. The signals responsible for these
chronic 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, and
the 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 AT
1
/AT
2
, and the ACE genotype are all variables that could impact the vascular responses to exercise
training, but the responses of most of these variables to regular exercise training and the mechanisms responsible have not
been systematically studied.
Key words: vasomotor function, endothelium, vascular smooth muscle, exercise, angiotensin-converting enzyme, oxidative
stress, ACE genotype, vascular health, hypertension.
Resume : Le syste`me renine-angiotensine (RAS) joue un role important dans la regulation de la pression sanguine et du
volume de liquide extracellulaire. Traditionnellement considere comme partie integrante du syste`me endocrinien, le
concept du RAS a evolue de telle sorte quon lui reconna t maintenant une fonction paracrine a` action locale comme on
lobserve dans les parois des vaisseaux sanguins. Langiotensine II (AII), leffecteur propre du RAS constitue un puissant
vasoconstricteur obtenu par laction de lenzyme de conversion de langiotensine (ACE). LACE a plusieurs fonctions
dont celle de detruire la bradykinine, une substance endoge`ne vasodilatatrice. LACE2, une enzyme recemment identifiee,
catalyse a` partir de lAII la formation de langiotensine 17 a` action vasodilatatrice. Ainsi donc, les actions de lACE et
de lACE2 sont antagonistes. Les actions tissulaires de lAII sont mediees par des recepteurs specifiques, AT1 et AT2, le
premier exercant son action classique. La vasoconstriction issue des AT1 depend de laction dun deuxie`me messager, la
phospholipase D, en ce qui concerne la voie directe et du couplage de AT
1
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 despe`ces re-
actives oxygenees dans les vaisseaux et, en outre, catalyse la degradation de loxyde nitrique (NO), une substance
vasodilatatrice, 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. Rush
1
and C.D. Aultman. Department of Kinesiology, University of Waterloo, Waterloo, ON N2L 3G1, Canada.
1
Corresponding author (e-mail: jwerush@uwaterloo.ca).
162
Appl. Physiol. Nutr. Metab. 33: 162172 (2008) doi:10.1139/H07-147 # 2007 NRC Canada
par les recepteurs AT2 sont opposes a` ceux des recepteurs AT1 et concourent a` la formation de bradykinine et de NO.
Laction chronique de AII entra ne un mauvais fonctionnement des vaisseaux sanguins et lintervention pharmacologique
aupre`s du RAS contribue non seulement a` la regulation de la pression sanguine, mais a` corriger les fonctions endotheliales
chez les hypertendus. Lentra nement physique peut aussi ameliorer les fonctions endotheliales chez les hypertendus, ce
qui veut dire que le RAS aurait peut-etre un role de mediation dans les adaptations des vaisseaux sanguins a` lentra -
nement physique. Des etudes recentes ont observe une diminution de lexpression des constituants de la NADPH oxydase
dans les parois des vaisseaux sanguins en reponse a` lentra nement physique, dou` lattenuation dun des effets principaux
de lAII dans la cellule et la diminution de la production de ROS dans les vaisseaux sanguins et une amelioration de la
biodisponibilite de NO. Tout aussi important, il est maintenant bien etabli que lentra nement physique attenue aussi lex-
pression des recepteurs AT1 dans les vaisseaux sanguins et la vasoconstriction causee par AII, ce qui ameliore la capacite
de vasodilatation de lendothelium. Les signaux responsables de ces adaptations chroniques ne sont pas bien compris et
semblent incorporer des modifications des constituants de RAS suscitees par lexercice physique. Le genotype de lACE
peut aussi avoir un effet sur les niveaux dactivite physique requis et sur les adaptations cardiovasculaires a` lentra nement
physique ; dans le genotype II (comparativement a` ID et DD), on observe une plus grande vasodilatation dorigine endo-
theliale chez les athle`tes que chez les sedentaires. En conclusion, la localisation de RAS, les actions combinees de lACE
et de lACE2, lexpression des recepteurs AT
1
et AT
2
de meme que le genotype de lACE sont toutes des variables qui in-
fluencent les adaptations vasculaires a` lentra nement physique, mais les adaptations de la plupart de ces variables a` len-
tra nement physique et les mecanismes responsables de ces adaptations nont pas fait lobjet detudes systematiques.
Mots-cles : fonction vasomotrice, endothelium, muscle lisse des vaisseaux sanguins, exercice, enzyme de conversion de
langiotensine, ECA, stress oxydatif, genotype, sante vasculaire, hypertension.
[Traduit par la Redaction]
______________________________________________________________________________________
Physiological biochemistry of the renin
angiotensin system
The reninangiotensin system (RAS) plays an essential
role 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 the
kidney in response to reductions in blood pressure, decreases
in distal tubule sodium chloride transport, and increases in
sympathetic tone. Renin cleaves angiotensinogen, an inac-
tive plasma protein synthesized in the liver, to form the de-
capeptide angiotensin I. Circulating angiotensin I (AI), in
turn, is cleaved to the octapeptide angiotensin II (AII) by an-
giotensin-converting enzyme (ACE; a dipeptidyl carboxy-
peptidase), found in plasma, abundantly in pulmonary and
vascular endothelial cells, and in the heart, kidneys, and
brain (Brewster and Perazella 2004; Jackson 2006; Siragy
1999) (Fig. 1). ACE exists at the cell surface as an ectoen-
zyme. The soluble (plasma) form of ACE results from the
action of ACE secretase, which cleaves the active portion
of the ACE protein from the membrane-bound portion (Hall
2003). AII is the main effector of the RAS, elevating arterial
pressure rapidly as a result of its powerful vasoconstrictor
action, 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 enzymes
in the tissue and blood inactivate AII.
Although the RAS was classically considered a circulating
system triggered by renin release and having general sys-
temic effects, it is now recognized that there are also local
tissue 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 of
RAS activation; the presence of local RAS allows tissue
AII concentrations to be much higher than circulating con-
centrations, and may place the sites of generation of AII in
closer proximity to the effector tissues expressing AII recep-
tors, i.e., the AII target organs (Siragy et al. 1995). Local
RAS further affects the pharmacological management of
this system because ACE inhibitors are more effective on
circulatory 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 AII
concentrations 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 by
non-ACE-dependent pathways, either from angiotensinogen
by the enzymes cathepsin G, elastase, and tissue plasmino-
gen activator, or from angiotensin I by enzymes, including
chymase (Fig. 1; Brewster and Perazella 2004; Hollenberg
et 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 during
ACE inhibitor therapy.
Another curiosity related to ACE inhibitor therapy results
from ACEs multifunctional enzyme activity. In addition to
its catalytic role in the formation of AII from AI, ACE also
cleaves and inactivates the naturally occurring vasodilators
bradykinin and kallidin (Fig. 1; Fleming et al. 2005; Yang
et al. 1970). Thus, inhibition of ACE is expected to not
only reduce AII levels and AII-induced vasoconstriction,
but also to potentiate bradykinin-induced vasodilation. The
latter effect may contribute to the observed antihypertensive
effects of ACE therapy.
Recent developments in ACE enzymology
Recently, 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 expressed
in vascular endothelial cells of the heart and kidney, and
can be released from the cell surface in a manner analogous
to the process for ACE (Donoghue et al. 2000; Tipnis et al.
Rush and Aultman 163
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2007 NRC Canada
2000). Although the general catalytic function of ACE2 is
similar to that of ACE, the specific petidyl cleavages cata-
lyzed by ACE2 are different from those of ACE, and include
cleavage of the carboxyl terminal phenylalanine residue
from AII to form the vasodilator peptide angiotensin-(17)
(Vickers et al. 2002). ACE2 may thus functionally antago-
nize the classic RAS by both removing AII and producing
angiotensin-(17). Furthermore, ACE2 is not sensitive to
ACE inhibitor drugs (Tipnis et al. 2000), and thus the in-
fluence of ACE2 would be synergistic to ACE inhibitor
therapy. Indeed, potential cardioprotective effects of ACE2
expression have been observed in heart development and
disease models (Crackower et al. 2002), but an integrative
appreciation of the vascular biology of ACE2 and its con-
tribution to control of vasomotor function has not been ex-
plored.
Vascular AII receptors and intracellular
signaling
The tissue actions of AII are mediated by two specific
cell-surface AII receptor types: AT
1
and AT
2
. Both receptor
types have seven transmembrane domains, and are G-protein
coupled, although some cellular actions elicited by AT acti-
Fig. 1. Schematic illustration of the reninangiotensin system (RAS) including tissue sites, enzymes, and intermediates, as well as functional
impacts on blood vessels. ACE, angiotensin-converting enzyme, AT1, angiotensin II recptor type 1. Bottom inset illustrates the coupling of
AT1 to NADPH oxidase in vascular cells and the potential relationship between AII signaling and the nitric-oxide-mediated, endothelium-
dependent dilation that this establishes.
164 Appl. Physiol. Nutr. Metab. Vol. 33, 2008
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2007 NRC Canada
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 AT
1
re-
ceptors (Allen et al. 2000). In addition to the renal effects
of increasing water and sodium retention (Goodfriend
2000), AT
1
receptor activation evokes G-protein-dependent
and -independent responses in the arterial vascular smooth
muscle (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 cell
growth (Wang et al. 2001), proliferation (Wolf and Wenzel
2004), and extracellular matrix formation (Otsuka et al.
1998). Since this review is focused on vasomotor effects of
AII, only the cell-signaling mechanisms pertinent to this
function will be discussed here. Other recent reviews contain
a broader perspective on AT receptor signaling and regula-
tion (Inagami et al. 1999; Kaschina and Unger 2003; Siragy
1999).
The constrictory effects of vascular smooth muscle AT
1
receptor activation occur through the activation of phospho-
lipase C (PLC), causing the hydrolysis of phosphotidylinosi-
tol 4,5-bisphosphate (PIP
2
) to form inositol 1,4,5-
trisphosphate (IP
3
) and diacylglycerol (DAG). IP
3
signaling
causes elevation in myoplasmic Ca
2+
, and DAG signaling
causes protein kinase C activation. Both signaling events
prompt VSM contraction. Synergistically, AT
1
-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 AT
1
activation
occur as a result of AII-induced augmentation of norepi-
nephrine (NE) release from sympathetic nerve terminals and
enhancement of the VSM contraction response to NE (Balt
et al. 2001), as well as AII-mediated expression of the
powerful vasoconstrictor endothelin-1 (Hahn et al. 1993).
AT
1
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 effect
of AT
1
receptor activation. The direct and indirect vasocon-
strictory effects of AT
1
activation are illustrated in Fig. 1.
While the AT
2
receptor is prevalent in fetal tissue, impli-
cating a possible role in growth and development, it is also
expressed 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 AT
2
receptor, it is
becoming apparent that AT
1
and AT
2
can have antagonistic
effects. For instance, with respect to vascular distribution
and vasomotor effects, some research suggests that the ef-
fects of AT
2
receptor activation promotes vasodilation and
antiproliferation (Siragy 1999; Siragy et al. 1999). There are
multiple intracellular signaling networks and functional out-
comes coupled to AT
2
receptors, and much of the detail has
not been well characterized. Part of the reason for the poor
characterization of the AT
2
receptor results from the more
limited distribution and density of AT
2
vs AT
1
and the an-
tagonistic interaction of these two receptors (reviewed in
Inagami et al. 1999; Kaschina and Unger 2003; Siragy
1999). The vasodilatory response to AT
2
activation has
been shown to depend on AT
2
- dependent bradykinin pro-
duction, which, in turn, results in BK
2
receptor activation,
eNOS activation, NO production, and NO-mediated dilation
through the cGMP mechanism in VSM (reviewed in
Kaschina and Unger 2003). This mechanism of AT
2
recep-
tor-mediated events has been confirmed in cultured endothe-
lial cells and in the aortas of stroke-prone spontaneously
hypertensive rats (Gohlke et al. 1998). To specifically inves-
tigate the AT
2
-receptor-mediated effects of AII, the AT
1
re-
ceptor must be blocked. The sartan drugs are angiotensin
receptor blockers (ARB) that specifically block the AT
1
re-
ceptors, whereas the experimental drug PD 123319 is a se-
lective ARB for the AT
2
receptor. These agents have been
useful not only clinically, but also experimentally in distin-
guishing mechanisms of action of AT
1
and AT
2
. The AII-
evoked elevation in cGMP was shown to be inhibited by
PD-123319, but not by losartan, confirming that this is an
AT
2
-mediated event (Siragy et al. 1996; Siragy and Carey
1999). The importance of this AT
2
effect is also supported
by gene manipulation studies in which AT
2
is either
knocked out or overexpressed. Knockout mice lacking AT
2
have greater vasoconstrictory responses to AII and elevated
blood pressure, whereas AT
2
overexpression induces vasodi-
lation and activation of the vascular kinin NOcGMP sys-
tem (Hein et al. 1995; Siragy et al. 1999; Tsutsumi et al.
1999). Furthermore, AT
2
receptor antagonism induces hy-
pertension (Siragy and Carey 1999). Together, these results
provide functional evidence that AT
2
mediates an important
vasodilatory role in adult animals. This could possibly be a
protective measure in normal blood pressure regulation and
vascular homeostasis, especially when the RAS is chroni-
cally activated, such as during hypertension and chronic
heart failure. A true balancing and antagonism between AT
1
and AT
2
actions seems to occur in terms of the vascular
vasomotor effects of AII (Inagami et al. 1999; Kaschina
and Unger 2003; Siragy 1999). Thus, a further character-
ization of AT
2
distribution and function in the adult
cardiovascular system is a necessary step toward a compre-
hensive understanding of the RAS.
Interaction of AII and nitric oxide-dependent
vasomotor activity: NADPH oxidase
ROS contribute to intracellular signaling maintenance of
vascular integrity (Touyz 2004). They are produced by multi-
ple enzymes, but quantitatively, NAD(P)H oxidase appears
to be the most significant source in the vasculature (Kojda
and 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)H
oxidase activation as a cause for reduced NO bioavailabil-
ity and accelerated endothelial dysfunction in hypertension
(Rajagopalan et al. 1996; Kerr et al. 1999; Graham and
Rush 2004; Rush et al. 2005) and other CV disease states
(Lassegue and Clempus 2003; Ohara et al. 1993; Kim et
al. 2002; Nedeljkovic et al. 2003). Thus, a possible site of
convergence linking endothelial dysfunction with AII-
dependent signaling processes is the regulation of NADPH
oxidase.
NAD(P)H oxidases comprise both membrane-bound and
cytosolic subunits that vary between vascular cell types. As-
suming functional assembly of subunits, activation of
Rush and Aultman 165
#
2007 NRC Canada
NADPH oxidase is induced by multiple mechanical and
chemical factors, including AII (De Keulenaer et al. 1998a;
Chen and Keaney 2004; Brandes and Kreuzer 2005; De
Keulenaer et al. 1998b; Griendling et al. 2000). AII-induced
ROS generation from NADPH oxidase is a biphasic re-
sponse (Seshiah et al. 2002; Laplante et al. 2005). The ini-
tial increase is initiated by AT
1
-dependent activation of
PLC, with DAG-dependent activation of PKC causing phos-
phorylation of the NADPH oxidase subunit p47phox, and
subsequent increases in O
2
.

. The second rise in ROS pro-

duction is PKC independent and involves activation of the
tyrosine kinase Src by H
2
O
2
, a O
2
.

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 phospholipase
A
2
(PLA
2
) increase formation of arachidonic acid (AA) and
its metabolic byproducts. Rac, AA, and leukotrienes can di-
rectly stimulate NAD(P)H oxidase (Brandes and Kreuzer
2005; Seshiah et al. 2002; Siragy 1999). It is notable that
this complex pathway creates a positive feedback loop initi-
ated by AII, in which O
2
.

enhances PKC activation, while

H
2
O
2
activates both Src and EGF, which in turn enhance
ROS 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 explain
why small changes in AII can result in large changes in vas-
omotor functional effects, including a dramatic rise in blood
pressure.
While few studies have examined endothelial function in
response to AII infusion, some attempts have been made
to elucidate the ROS and blood pressure-dependent and
-independent mechanisms responsible for altered vasomotor
function in models manipulating RAS and NADPH oxi-
dase. Interruption of the gp91phoxp47phox interaction
abolishes O
2
.

production and attenuates systolic blood

pressure in AII-infused mice (Rey et al. 2001), suggesting
a critical role for NADPH oxidase in the AII-mediated vas-
cular dysfunction and hypertensive effects. AII-induced
O
2
.

production is also inhibited in cultured endothelial

cells from p47phox knockout mice, and is restored in this
model by transfection of p47phox cDNA (Li and Shah
2003). It is thus clear that the effectiveness of AT
1
-receptor
inhibition for essential hypertension may be attributed, in
part, to reduced NAD(P)H oxidase activation and assem-
bly, leading to diminished ROS production and enhanced
NO bioavailability.
Using vasoconstrictors with different mechanisms of ac-
tion, Laursen et al. (1997) demonstrated a separation of
blood pressure effects from the endothelial dysfunction and
ROS production effects accompanying AII action. Thus,
whereas NE and AII infusion for 5 days caused a similar
increase in blood pressure, vascular O
2
.

production was in-

creased 3-fold by AII, but was not affected by NE, and en-
dothelium-dependent dilation was impaired only in the AII
group (Laursen et al. 1997). In vivo administration of lipo-
some-encapsulated SOD normalized O
2
.

production, parti-
ally restored endothelial-dependent relaxation, and partially
attenuated 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 brachial
artery dilation to Ach in healthy young men, acute infusion
of AII attenuated the ACh-induced dilatory response, and
the AII effect was abolished by simultaneous infusion of
the antioxidant vitamin C (Hirooka et al. 2003). Collec-
tively, these findings suggest both a pressure-independent
mechanism for endothelial dysfunction involving reduced
NO bioavailability, and a ROS- and NO-independent com-
ponent of AII-induced hypertension per se.
Pharmacological management of RAS and
vascular 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 and
AT
1
receptor blockers in attenuating blood pressure and en-
dothelial dysfunction is well documented (e.g., Azevedo et
al. 2003; Brosnan et al. 2002; Jackson 2006; Rodrigo et al.
1997; Romero and Reckelhoff 1999). Whereas the literature
indicates that individually both ACE inhibitors and AT
1
re-
ceptor blockers are effective in controlling hypertension, the
concomitant administration of both drug types elicits more
substantial improvements (Raasch et al. 2004) because of
distinct 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 AT
1
blockers are also present,
the persistent AII can interact with AT
2
receptors and elicit
a significant vasodilation; and second, bradykinin is a potent
vasodilator through the BK
2
receptor coupled to eNOS acti-
vation in vascular endothelium. These responses may oppose
AT
1
-mediated constriction, thus contributing to the effec-
tiveness these drugs. In contrast to ACE inhibitors, AT
1
receptor blockers do not evoke the same beneficial increase
in bradykinin production, but they inhibit AT
1
-receptor-
mediated responses and enhance AT
2
activation by AII.
Although plasma AII concentration is elevated in disease
states, including renovascular hypertension (Simon and
Abraham 1995) and chronic heart failure (Brink et al.
1996), more than 50% of people with essential hypertension
have plasma AII levels comparable to those of normotensive
people (Romero and Reckelhoff 1999), and hypertensive rat
models 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 AT
1
receptor blockers reduce blood pressure
and 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 this
apparent paradox may lie in appreciating that, although
plasma AII levels are not elevated above normal values
found in normotensive individuals, they are also not lowered
by the prevailing cardiovascular conditions (e.g., increased
mean arterial pressure should lower renin release, RAS acti-
vation, and AII production). The observation that this does not
occur in the cases mentioned prevents the re-establishment
of cardiovascular homeostasis and probably accounts for
the paradoxical findings (Simon and Abraham 1995;
Romero and Reckelhoff 2000). A potential mechanism pro-
posed to explain how near-normal plasma levels of AII
maintain chronic elevations in blood pressure is AII-in-
166 Appl. Physiol. Nutr. Metab. Vol. 33, 2008
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2007 NRC Canada
duced activation of NAD(P)H oxidase, as outlined above,
and enhancement of its expression (Seshiah et al. 2002).
Thus, because of AIIs various mechanisms of action, the
history of its exposure may affect the vascular biological
responses on different time scales and with different appa-
rent sensitivities.
Chronic effects of AII on vascular phenotype
Chronic in vivo AII infusion also increases the expression
levels of NAD(P)H oxidase subunits in both mouse and rat
aorta (Cifuentes et al. 2000; Mollnau et al. 2002), and these
increases are attenuated by chronic administration of the
AT
1
receptor blocker irbesartan (Brosnan et al. 2002). Con-
sistent with reported impairments in endothelial-independent
dilation, chronic treatment with AII also reduces soluble
guanylate cyclase (sGC) expression in rats, implying re-
duced VSM sensitivity to NO (Laursen et al. 1997; Mollnau
et al. 2002). Chronic AII infusion also increases eNOS
protein 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 superoxide
dismutase (ecSOD) are also increased in AII- but not NE-
infused mice, implying a pressure-independent mechanism
of 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). Increased
ecSOD may preserve NO in the extracellular space through
which it must diffuse. Concomitant administration of the
AT
1
receptor blocker losartan abolishes all AII-induced
changes in ecSOD. Thus, there is growing evidence that
chronic 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 critical
roles in normal physiological states, the antagonistic interac-
tion of NO with AII and ROS demands an intricate balance
to maintain cardiovascular homeostasis (Schulman et al.
2005). When the balance is disrupted, vascular dysfunction
results. Chronic exercise training is a known stimulus for
vascular adaptations and has been shown to improve im-
paired vasomotor function (Rush et al. 2005; Kojda and
Hambrecht 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 CV
homeostasis.
Angiotensin system and vascular
adaptations to chronic exercise training
Exercise training elicits improvements in endothelial-
dependent dilation in healthy controls and in a variety of
CVD models, including normotensive (Delp et al. 1993)
and hypertensive rats (Graham and Rush 2004), hypercho-
lesterolemic pigs (Thompson et al. 2004), and humans with
essential hypertension (Higashi et al. 1999), chronic heart
failure (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 activity
of pathways influencing NO bioavailability (Kojda and
Hambrecht 2005; Rush et al. 2000, 2005). Exercise training
for 10 weeks also reduced the sensitivity of abdominal
aortic rings to KCl-induced contraction by an endothelial-
independent mechanism (Delp et al. 1993), and to
NE-induced contraction by an endothelial-dependent mech-
anism involving a
2
-adrenergic receptors (Spier et al. 1999;
Delp et al. 1993). These findings lend potential insight into
another possible mechanism of exercise training-induced
enhancements in NO bioavailability, because NE binding
to endothelial a
2
-adrenergic receptors increases intracellular
Ca
2+
, 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 exercise
specifically on the vascular RAS or its role in vasodilatory
or vasoconstrictory adaptations to exercise training.
In addition to exercise training-dependent increases in the
expression and activity of vascular eNOS and antioxidant
enzymes that favor enhanced NO bioavailability (Kojda and
Hambrecht 2005; Rush et al. 2005), growing evidence sup-
ports the possibility that exercise induces adaptations in
NADPH oxidase (Adams et al. 2005; Graham and Rush
2004; Kojda and Hambrecht 2005; Rush et al. 2003, 2005),
which, because of its importance as an effector of the AII
signaling system, could interface the RAS with the vascular
functional 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 in
rat thoracic aorta (Graham and Rush 2004) after chronic
aerobic exercise training of moderate intensity. Diminished
mRNA expression of gp91phox, Nox4, and p22phox, as
well as decreased protein expression of gp91phox in human
mammary arteries are also found following 4 weeks of train-
ing, and these changes are accompanied by attenuated
NAD(P)H oxidase activity and ROS production (Adams et
al. 2005). In the latter study, the reduction in NADPH oxi-
dase components was accompanied by decreases in vascular
wall NADPH oxidase activity and ROS production (Adams
et al. 2005). Thus, as NADPH oxidase can be considered
one of the main effectors of AII in the vascular tissue, this
effect is significant in terms of the potential role of the
RAS in the exercise training response. Indeed, the dilation
of mammary artery segments to acetylcholine was enhanced
in the trained vs the untrained group, and conversely, train-
ing blunted the contractile response to AII in vitro (Adams
et al. 2005).
As a possible explanation for the reduced maximal
AII-induced vasoconstriction in human mammary arteries
from coronary artery disease patients following training,
Adams et al. (2005) reported decreased AT
1
receptor and
increased AT
2
receptor mRNA in the mammary arteries of
trained vs untrained individuals. Whether coupled to
NADPH oxidase or to the PLA
2
mechanism of action, re-
duced AT
1
receptor expression would favor a blunted vaso-
constrictor response to AII, consistent with the results
presented (Adams et al. 2005). The universality of the ob-
served exercise effect on vascular AT receptor expression
will have to be assessed in various vascular beds and vessel
Rush and Aultman 167
#
2007 NRC Canada
types to fully evaluate the potential role of this adaptation
in the mechanism of improved vascular function and re-
duced vascular oxidative stress following exercise training.
Vascular adaptation to exercise may depend in part on the
ACE 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 specific
genetic factor associated with physical activity levels in
free-living individuals, with sedentary lifestyle more com-
mon among DD individuals than in II genotype borderline
and 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 in
aerobic power as a result of physical training in cardiac re-
habilitation than those that occurred in patients with the ID
or DD genotypes (Defoor et al. 2006). In the context of vas-
cular function and exercise, a comparison of athletes with
sedentary individuals within each genotype demonstrated
that athletes with the II ACE genotype had the greatest in-
creases in endothelium-dependent flow-mediated dilation of
the brachial artery, athletes with the DD ACE genotype had
the smallest enhancement of flow mediated dilation, and the
ID heterozygotes had an intermediate response (Tanriverdi
et al. 2005). Whether this reflects a contributing role for
ACE or angiotensin to the exercise training response in the
function of the vascular endothelium, or reflects an unknown
mechanism indirectly affiliated with the ACE genotype is
unknown. A systematic study of the influence of the ACE
genotype on the trainability of the endothelium and the
mechanisms responsible would make a significant contribu-
tion to the understanding of the mechanisms by which the
RAS influences cardiovascular adaptations to exercise.
A variety of physical and chemical signals associated with
exercise may prompt the molecular phenotypic changes that
in 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 studies
have confirmed that some of the effects of AII exposure are
similar to responses prompted by exercise training, such as
enhanced vascular eNOS and ecSOD expression (Fukai et
al. 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 levels
have been performed on subjects with a variety of compli-
cating conditions, in general, their results all demonstrate
that 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 may
not (Aldigier et al. 1993; Miura et al. 1994) be ACE
dependent. 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 within
10 min of beginning 70% VO
2 max
cycling exercise and it
continued 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 levels
for at least 40 min of recovery (Woods et al. 2004). The
persistent elevation of AII during and after acute exercise in
this study suggests that this molecule could influence vascu-
lar molecular phenotype and function via known effects of
AII (Fukai et al. 1999; Hennington et al. 1998; Moreno et
al. 2002). Acute exercise and exercise training studies using
ARB and (or) ACE inhibitors would be useful in testing this
hypothesis that AII dynamics during acute exercise bouts
contribute to exercise training-induced vascular phenotypic
adaptations. Whether these adaptations would occur via a di-
rect effect of AII signaling on gene expression, or indirectly
through AII-induced ROS, could be determined with antiox-
idant manipulations or NADPH knockout animals under-
going exercise protocols. However, because both basal
levels and the degree of acute exercise-induced elevations
in AII were independent of the ACE ID genotype in several
studies (Chadwick et al. 1997; Danser et al. 1999; Woods et
al. 2004), it is not likely that the hypothesized role for AII
could account for the genotype-dependent improvements in
endothelium-dependent flow-mediated dilation in athletes
compared 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 limited
to location, as there are multiple forms of carboxypeptidases
with 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 receptors
also have antagonistic effects, so the expression pattern of
these receptors in the tissue of interest, and any changes in
the 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 AT
1
receptor 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 NADPH
ROS production; and blunted expression of AT
1
in arteries
after 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, as
this molecule responds to acute exercise bouts and is known
to cause gene expression changes in vascular cells that, in
some cases, are similar to those resulting from exercise
training. 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 vascular
function justifies efforts to fully describe the vascular RAS
alterations with exercise training and determine their impor-
tance to the establishment of the functional adaptations of
168 Appl. Physiol. Nutr. Metab. Vol. 33, 2008
#
2007 NRC Canada
arteries to exercise that underlie changes in vasomotor activ-
ity, regulation of blood flow, and blood vessel structure.
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