Hypertension, RAS, and gender: what is the roleof aminopeptidases?

Marıa Jesus Ramırez-Exposito Æ Jose Manuel Martınez-Martos

Published online: 24 January 2008

� Springer Science+Business Media, LLC 2008

Abstract Hypertension is the major risk factor for coro-

nary heart disease, stroke, and renal disease. Also, it is

probably the most important risk factor for peripheral

vascular disease and vascular dementia. Although hyper-

tension occurs in both men and women, gender differences

have been observed. However, whether sex hormones are

responsible for the observed gender-associated differences

in arterial blood pressure, and which is their mechanism of

action, remains unclear. Local and circulating renin–

angiotensin systems (RAS) are examples of systems that

may be involved in the pathogenesis of hypertension.

Classically, angiotensin II (Ang II) has been considered as

the effector peptide of the RAS, but Ang II is not the only

active peptide. Several of its degradation products,

including angiotensin III (Ang III) and angiotensin IV (Ang

IV) also possess biological functions. These peptides are

formed via the activity of several aminopeptidases. This

review will briefly summarize what is known about gender

differences in RAS-regulating aminopeptidase activities,

their relationship with sex hormones, and their potential

role in controlling blood pressure acting through local and

circulating RAS.

Keywords RAS � Angiotensinase � Gender differences �Sex hormones

Introduction

Hypertension, defined traditionally as persistent blood

pressure, is one of the major contributors to premature

morbidity and mortality in the United States [1–3]. How-

ever, by the year 2025, hypertension is expected to increase

in prevalence by 60%, affecting 1.56 billion people [4].

Hypertension is a major risk factor for coronary heart

disease, the number one cause of death in the US [5], stroke

[6], which ranked number three as a cause of death in the

US in 2003, and renal disease. After smoking, hypertension

is probably the most important risk factor for peripheral

vascular disease [6]. Hypertension is also the most

important treatable cause of vascular dementia [5] (for

more data see review [3]).

Actually, between a fifth and a quarter of the adult

population would meet the criteria for hypertension [7].

The incidence of hypertension becomes more prevalent

with age [8] and hypertension is found in about 50%

of individuals above 55 years in many industrialized

countries.

Hypertension is a major problem for both men and

women. But men tend to develop it at an earlier age. Thus,

using the technique of 24-h ambulatory blood pressure

monitoring, it has been confirmed that blood pressure is

higher in men than in premenopausal women of similar

ages [9–11]. Gender differences in blood pressure are also

present in several hypertensive rat models such as spon-

taneously hypertensive rats (SHR) [12–15], Dahl salt-

sensitive rats [16, 17], deoxycorticosterone acetate-salt

hypertensive rats [18], and New Zealand genetically

hypertensive rats [19], where males have higher blood

pressure than do females [20]. However, in population

older than 60–70 years, women are more likely to have

hypertension [21, 22] and to suffer cardiovascular death,

M. J. Ramırez-Exposito (&) � J. M. Martınez-Martos

Experimental and Clinical Physiopathology Research Group,

Department of Health Sciences/Physiology, Faculty of

Experimental and Health Sciences, University of Jaen,

Campus Universitario Las Lagunillas, 23071 Jaen, Spain

e-mail: mramirez@ujaen.es

123

Heart Fail Rev (2008) 13:355–365

DOI 10.1007/s10741-008-9082-1

myocardial infarction, and heart failure than men [6].

Menopause’s contribution to this phenomenon is complex.

Estrogen deficiency after menopause precipitates a number

of factors and these have established the menopausal

metabolic syndrome as a concept in postmenopausal

women. However, studies have indicated that changes in

the prevalence of hypertension, and overall cardiovascular

risk profiles in postmenopausal women, might be due to

ageing and not estrogen deficiency [23].

The renin–angiotensin system (RAS)

Since Braun-Menendez et al. [24] and Page and Helmer

[25] isolated the octapeptide angiotensin II (Ang II), the

peripheral RAS was subsequently characterized. However,

the RAS of renal origin has undergone important recon-

siderations. In the same time with the identification of the

enzyme cascade that produces active Ang II, sufficient

experimental evidence has accumulated in favor of the

presence of all RAS components within most tissues and

organs [26]. Thus, the notion has evolved of an extra renal

RAS and the concept of circulating and tissue RAS as a

unitary hormonal system. On the other hand, it has been

observed that Ang II is not the only active component of

the RAS. New active angiotensin components were iden-

tified as metabolic products of Ang II, with similar or

different functional properties. Similar physiopharmaco-

logical properties were shown for the heptapeptide

angiotensin III (Ang III) [27–33], acting through common

receptors with Ang II, i.e., the AT1 or AT2 receptor sub-

types. Different properties were also demonstrated for the

hexapeptide angiotensin IV (Ang IV) [28–33] and hepta-

peptide angiotensin 1–7 with the involvement of specific

receptors. Considering the angiotensin-forming cascade,

the first enzyme involved is an acid aspartate-protease

called renin (EC 3.4.23.15), of renal origin [34], which

converts angiotensinogen to angiotensin I (Ang I). Ang I is

inactive, and converted to active Ang II by the dipept-

idylcarboxypeptidase angiotensin-converting enzyme

(ACE; EC 3.4.15.1) [35]. Through similar mechanisms

active Ang III and Ang IV are produced via the involve-

ment of additional angiotensinases.

Ultimately the physiological properties of bioactive

angiotensins depend upon their interaction with specific

membrane receptors, the true information transducers at the

cell level. Unlike circulating angiotensins of renal origin,

tissue-generated angiotensins have local hormonal proper-

ties of paracrine, autocrine, or intracrine type. Similar to

circulating angiotensins, they act via transmembrane

receptors, coupled with intracellular G protein or tyrosin-

ekinase. The specific receptors are represented by the AT1

and AT2 receptors, with their subtypes AT1A and AT1B for

Ang II and Ang III, and by the AT4 and AT1–7 receptors for

Ang IV and Ang 1–7, respectively. AT3 receptors have

been identified, but only in the neuroblastoma cell line

[36].

The prevalence of the AT1 receptors both in the various

peripheral tissues and organs and in certain central nervous

structures insures most of the classical actions of Ang II,

including its trophic and hyperplasic cardiovascular effects.

In contrast, AT2 receptors possess antagonistic properties

as related to the control of local blood flow via vasodilating

factors [37]. Unlike these, AT4 receptors predominate

within the cerebral cortex, hippocampus, and basal nuclei,

contributing to learning and memory processes [38]. The

activation of AT1–7 receptors induces vasodilation, divre-

sis, apoptosis, and antiproliferative actions.

According to the type and reactivity of each receptor

when activated by bioactive angiotensins, produce specific

actions. In the case of Ang II a great deal of experimental

evidence supports classic pressor, hydroelectrolytic, dips-

ogenic, vasopressin, calcium, and aldosterone-releasing

properties. However, this octapeptide also participates

through the AT1 receptors as a trophic and stimulating

factor of oxidative stress, inflammation, atherogenesis, and

cardiovascular modeling [39]. In addition to its multiple

own actions, many of these functions are also mediated by

active fragments of Ang II including Ang III, Ang IV, and

Ang 1–7 [40].

While Ang III induces, pressor, dipsogenic, and vaso-

pressin-releasing effects similar to Ang II, contributing to

the feeding dipsogenic behavior, Ang IV is a vasodilator

and stimulates the nervous processes of learning and

memory, accompanied electrically, by long-term potentia-

tion [41]. Intracerebroventricular administration of Ang IV

facilitates the conditioned reflexes of avoidance, and

potentiates the associative memory in rat through the

activation of acetylcholine release and modulation of

NMDA receptors [41]. The deficit of Ang IV production

due to the deterioration of the angiotensin-forming

enzymes induced by genetic causes, age, vascular, or

metabolic risk factors, might contribute to the progressive

neuronal degradation in Alzheimer disease. In favor of

cerebral RAS implications in the plurifactorial pathogen-

esis of Alzheimer disease plead both the decrease of the

ACE activity in the elderly and its participation in the

degradation of neurotoxic beta-amyloid protein from

the senile plaque [42]. It should be noted that a reduction of

ACE activity also results in a deficit of synthesis of Ang IV

precursors, contributing to progressive alterations in the

electrochemical processes of learning and memory [43].

Unlike the mainly central actions of Ang III and Ang IV,

Ang 1–7 produces peripheral vasodilator, diuretic, and

antiproliferative reactions through the release of nitric

oxide, kinins, and prostaglandins [44]. Ang 1–7, by

356 Heart Fail Rev (2008) 13:355–365

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inhibiting the presser and proliferative effects of Ang II,

behaves as a true factor for counteracting it, in order to

re-establish and maintain the basal vascular tone. Consis-

tent with these results is the hypothesis concerning a

possible balance between the vasoconstricting and hyper-

tensive actions of Ang II and the vasodilator properties of

Ang 1–7, as a genuine system for braking, modeling, and

self-regulation of the vascular actions of RAS [45].

Angiotensinases

Aminopeptidase (AP) activity plays a major role in the

metabolism of circulating and local peptides involved in

blood pressure and water balance. In this way, angioten-

sinases are peptidases that generate active or inactive

angiotensin peptides that alter the ratios between their

bioactive forms. Included in this category are aspartyl

aminopeptidase (AspAP) and glutamyl aminopeptidase

(GluAP), named together as aminopeptidase A (APA) (EC

3.4.11.7), aminopeptidase B (APB) (EC 3.4.11.8), and

aminopeptidase N (APN) (EC 3.4.11.2), involved in the

removal of the first amino acids of the peptide chain of Ang

II for the formation of Ang III and Ang IV. While APA and

APB and APN successively remove the first and the second

amino acids of the peptide chain of Ang II, producing Ang

III and Ang IV. In their turn, tissue carboxypeptidases and

other proteolytic enzymes (such as trypsin, chymotrypsin,

pepsin, and others) contribute to the inactivation of the

active forms of angiotensins transforming them into inac-

tive fragments and amino acid constituents [35, 46–48]. A

separate pathway for the synthesis of Ang III that does not

depend upon the formation of Ang II is via the nonapeptide

[des-Asp1]AngI, formed from Ang I by AspAP [49]. This

nonapeptide is converted directly to Ang III via ACE. Sim

and Radhakrishnan [50] reported that intracerebroventric-

ular infusion of [des-Asp1]AngI produced an elevation in

blood pressure in SHR and Wistar-Kyoto (WKY) normo-

tensive rats. This indicates that [des-Asp1] must be

converted to Ang III in order for biological activity to

occur.

Aminopeptidase A

Aminopeptidase A appears to be a membrane-bound

enzyme widely distributed in the body, with especially high

levels in the kidney [51]. Although this activity is also

present in vascular circulation, its origin is not totally

understood. It has been suggested that this enzyme

becomes soluble in plasma via autolysis of membrane-

bound APA [52]. The only aminopeptidase previously

reported to be specific for dicarboxylic amino acids is

glutamate aminopeptidase (GluAP) which readily removes

the N-terminal Asp residue from L-Asp1-Ang II [53].

GluAP does not hydrolyze 2-naphthylamide derivatives of

amino acids other than aspartic and glutamic acids. This

specificity also applies to the hydrolysis of true peptides

bonds such as those in angiotensin analogs [54]. An

aspartate aminopeptidase with low activity on 2-naph-

thylamide derivatives may also be partially responsible for

the hydrolysis on N-terminal Asp residues of peptides.

Therefore, with AspNNap as the substrate, the release of

2-naphthylamine may be due to the action of at least two

different enzyme activities (APA activity). Because of its

specificity and its rapid action on N-terminal aspartic acid

residues of angiotensin analogs, AspNNap hydrolyzing

activity may also act as an angiotensinase [54].

Gender differences and angiotensinase activity

Ten years ago, we performed in our lab, the first experi-

ments to analyze the relationship between gender and

hypertension. Our group demonstrated a different profile

for GluAP and AspAP activities in males and females,

which supports the existence of two enzymes with pre-

sumably different properties and roles in the regulation of

susceptible substrates [55]. We evaluated the activity of

APA by determination of GluAP and AspAP in human

serum during development and ageing, in an apparently

healthy population of 139 male and 148 female subjects.

We found sex differences and also age-related changes in

APA activity [55]. For GluAp we observed a progressive

increase during development and ageing, which was more

prominent in females. The profile of AspAP activity was

also different in females and males, essentially after

45 years of age. Sanderik et al. [56] reported higher values

for Alanyl aminopeptidase activity (AlaAP) in males than

in females, and also noted sex differences during devel-

opment, i.e., an inverse correlation with age in males (10%)

and high levels in females (15%) among hypertensive

subjects. AlaAP (APN; EC 3.4.11.2) has also been pro-

posed as an angiotensinase [52]. However, our results

suggested a more specific action of APA, particularly

GluAP, in the metabolism of circulating Ang II during

development and ageing. Interestingly, AspAp activity did

not correlate significantly with age or with sex [55]. Blood

pressure, particularly systolic blood pressure, tends to

increase progressively with age [57–59]. Blood pressure

increases more in males than in females, and values in

elderly males and females are similar. In the RAS, plasma

renin activity and Ang II decline progressively with age

[58–62]. The decrease in plasma renin activity is expo-

nential, being very rapid in early childhood and continuing

into old age. The contractile response to Ang II decrease

Heart Fail Rev (2008) 13:355–365 357

123

also with age [63]. This age-related profile is the opposite

of the one found in our study for APA activity. Therefore,

the inverse correlation between peptide level and peptide-

hydrolyzing activity suggested a relation between high

breakdown capacity and low level of substrate, which in

turn supports a role for human plasma APA activity as an

angiotensinase. However, these observations appear

incompatible with the above-mentioned tendency of blood

pressure to increase with age and for Ang II-induced

contractility to decline. Therefore, additional factors must

be involved in this phenomenon which could include

changes with ageing that results in gradually increasing

rigidity of vessels. In addition, the existence of sex dif-

ferences and the fact that the most significant changes were

observed after puberty and elderly subjects suggest that

hormonal status is involved in APA activity, which is also

in agreement with the results of Sanderink et al. [56] for

human serum AlaAP activity. Moreover, previously the

influence of sexual hormones on AP activities was deter-

mined. In fact, the involvement of the hormonal status on

aminopeptidase activity was also in agreement with other

in vitro results which demonstrated a direct influence of

steroid hormones on aminopeptidase activities, especially

on GluAP and AspAP [64].

The mechanisms responsible for sex differences in

blood pressure control and regulation are not fully under-

stood, but appear to involve effects of sex hormones [65].

A rightward shift of the pressure–natriuresis curve with a

resultant long-term increase in blood pressure has been

reported in male animal models of hypertension. Castration

restores the pressure–natriuresis relationship, and androgen

receptor blockade lowers blood pressure in these models,

providing evidence that androgens contribute to the higher

blood pressure observed in males [66]. Administration of

testosterone to ovariectomized SHR rats has been shown to

elevate blood pressure and blunt the pressure–natriuresis

relationship, suggesting the possibility, as yet untested in

clinical studies, that androgens may play a role in the rise

in blood pressure that occurs in menopausal women.

Female sex hormones, in contrast, appear to protect against

salt-induced increases in blood pressure, at least in part by

increasing sensitivity of the pressure–natriuresis relation-

ship and augmenting renal excretion of sodium [67].

Studies in animal models have revealed emergence of salt-

sensitive hypertension after ovariectomy. Activation of the

sympathetic nervous system, alterations in salt appetite,

and modulation of many functions of the renin–angioten-

sin–aldosterone system appear to participate in the

pathogenesis of salt-sensitive hypertension in these models.

Additional mechanisms that have been suggested to

explain the effects of ovarian hormones on blood pressure

include the maintenance of normal endothelial function

[68], reductions in plasma renin activity, angiotensin-

converting enzyme activity, and vascular AT1 receptor

expression and also induction of structural and functional

alterations in the arterial wall [65].

In this way and with these premises, we designed a new

study in which we analyzed the effects of orchiectomy and

ovariectomy on aminopeptidases involved in the metabo-

lism of active peptides of systemic and tissular RAS.

As we mentioned before, in addition to the circulating

RAS, local systems have been postulated in brain, pituitary,

and adrenal glands [69–72]. These local RAS have been

implicated, respectively, in the central regulation of the

cardiovascular system and body water balance, the secre-

tion of pituitary hormones [73] and the secretion of

aldosterone by adrenal glands [74]. On the other hand, it is

known that the hypothalamus–pituitary–adrenal (HPA)

axis is involved in blood pressure regulation and is affected

by sex hormones. In our lab, we analyzed the influence of

testosterone on RAS-regulating APA, APB, and APN

activities in the HPA axis, measuring these activities in

their soluble and membrane forms in the hypothalamus,

pituitary, and adrenal glands of orchiectomized males and

orchiectomized males treated subcutaneously with several

doses of testosterone. We also analyzed vasopressin-

degrading cystyl-aminopeptidase activity (AVP-DA) (EC

3.4.11.3) [75] because it is known that Ang III is particu-

larly important in brain and especially in pituitary

physiology and plays a major role in the secretion of

arginine-vasopressin (AVP) [76]. AVP is also implicated in

the regulation of blood pressure [77] and is metabolized by

AVP-DA. Those data suggested that in male mice, testos-

terone influences the RAS- and AVP-regulating activities

at all levels of the HPA axis [78]. Thus, orchiectomy

modified hypothalamus APA, APN, and APB; pituitary

APN and APB; and adrenal APA, APN, and APB and

AVP-DA, whereas testosterone replacement return the

activity to control or under control levels, depending on

the dose used. We also described, that the administration of

the highest doses of testosterone modified some activities

which were not altered by orchiectomy, probably due to

pharmacological rather than physiological effects. This

presumable pharmacological effect also appears when the

highest doses of testosterone were used under condition in

which lower doses return the activity to control levels after

being modified by orchiectomy.

With regard to the different levels of the HPA axis, it is

well known that several hypothalamic nuclei are involved

in neuroendocrine regulation through RAS action. Thus,

in vivo administration of Ang II appears to act by stimu-

lating CRH secretion [79]. Furthermore, Ang III behaves as

one of the main effector peptide of the RAS in the control

of AVP release [80, 81]. In fact, Ang III possesses most of

the properties of Ang II and may also activate AT1 and

AT2 receptors [76, 82]. Our data may indicate that, under

358 Heart Fail Rev (2008) 13:355–365

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androgen deprivation, Ang II is converted into Ang III by

APA more rapidly in hypothalamus, being Ang III also

quickly converted in Ang IV by APB and APN. Although

Ang IV has been implicated mainly in memory and

cognitive processes and sensory and motor integration

[73], it has been also described that Ang IV produces

opposing responses to Ang II with respect to local tissue

blood flow, regulating in certain degree, Ang II/Ang III

functions [83]. Therefore, the functions of Ang II and

Ang III may be diminished and blunt the release of CRH

and AVP from hypothalamus. In fact, long-term orchi-

ectomy decreased hypothalamic CRH content [84].

However, several studies suggest that the effects of

androgen in the CNS to augment HPA axis activity are in

part secondary to an interaction with corticosteroid neg-

ative feedback mechanism. In males, orchiectomy

increased ACTH responses whereas androgen treatment

decreased ACTH response [85]. Testosterone replace-

ment, however, appeared to decrease AVP content in the

median eminence [86]. However, the effect on AVP

mRNA was observed as an interaction with adrenal ste-

roids [87]. Taken together, the results suggested that

androgens interact with adrenal steroid in regulation of

AVP synthesis in the hypothalamus. These results also

suggested that the site of steroid hormone action is the

hypothalamus rather than the pituitary.

In the pituitary, RAS, Ang II stimulates in a paracrine

manner the secretion of adrenocorticotrophin (ACTH) [88].

Our data may indicate that androgen deprivation did not

change APA activity, but induced the conversion of Ang III

to Ang IV more rapidly, maintaining the levels of Ang II,

which could be also regulated by Ang IV, helping to

maintain low levels of ACTH. In this way, Lesniewska

et al. [89] had described that orchiectomy decreases pitu-

itary ACTH levels in rats. These data may indicate that low

levels of glucocorticoids will be present in castrated males.

In adrenal glands, there is little doubt that both circu-

lating and intra-adrenal RAS can stimulate aldosterone

secretion, being Ang II the main regulator of its secretion

in the adult animals [90]. Our data showed the same pattern

of RAS-regulating aminopeptidases than in hypothalamus.

Thus Ang II may be converted into Ang III by APA more

rapidly and then quickly converted into Ang IV by APB

and APN, which could be responsible of low aldosterone

levels. In any case, there may be other functions of the

intra-adrenal RAS apart from these direct effects on aldo-

sterone production, involved in the local regulation of

adrenal blood flow, which must been taken into account. In

view of these results we can conclude that the increasing

effects of testosterone in blood pressure may be mediated

by changes in the RAS-regulating aminopeptidases activi-

ties at different levels of the HPA-axis and AVP-DA in

adrenal glands [78].

Similar experiments were designed to analyze the

influence of estrogen at the same levels. We observed

changes in GluAP and AspAP activities in hypothalamus,

pituitary APB and GluAP, APB and APN in adrenal glands

after ovariectomy, but the replacement of estrogen gener-

ally returned the activities to control levels. Our data may

indicate that, under estrogen deprivation, Ang II is con-

verted into Ang III by Glu and AspAP more rapidly in

hypothalamus, being Ang III increased because APB and

APN did not change. In pituitary, the changes observed in

the AP activities demonstrated that Ang III was decreased

and Ang IV increased and finally in adrenal gland the

levels of Ang III may also be higher because the metabo-

lism of Ang II is increased. Whereas testosterone can act to

inhibit HPA functions, estrogen can enhance HPA function

[85, 91]. In both sexes estrogen administration increases

basal corticosterone secretion as well as ACTH and corti-

costerone response to physical and psychological stressors

[85]. Further support for estrogen altering HPA function is

demonstrated by studies examining basal and stress-

responsive corticosterone and ACTH release across the

estrous cycle. On proestrus (high circulating estrogen lev-

els) corticosterone levels are greatest [92]. The effect of

estrogen secretion has been studied [93] suggesting that

one mechanism by which estrogen enhances stress hor-

mone secretion is by impairing glucocorticoid receptor-

mediated negative feedback. Along with the changes in

hormone secretion, other studies had also demonstrated

changes in CRH levels and CRH mRNA within the para-

ventricular nucleus in the presence of estrogen [85, 94]. In

addition to these centrally mediated effects of estrogen, it

has been reported that estrogen can increase corticosterone

production by the adrenal gland [95] and increase anterior

pituitary sensitivity to CRH-containing hypothalamic

extract [96]. These classic studies suggested that the

addition to changes in neuronal function, estrogen can

modulate the HPA axis [85].

The activation of the HPA axis by angiotensins may

contribute to the pathogenesis of hypertension [97]. In this

way, Hinojosa-Laborde et al. [98] had previously showed

that ovariectomy of adult (4 months) normotensive female

rats induced spontaneous hypertension. On the other hand,

estradiol (E2) replacement significantly reduced blood

pressure in ovariectomized rats through 12 months of age

[98]. These authors demonstrated that the ovarian hormone,

estrogen, plays a key role in the ovariectomized-induced

hypertension. Ovariectomy also decreased progesterone (P)

levels but this hormone had no effects on the development

of hypertension in ovariectomized rats [99]. Estrogen has

been shown to suppress the RAS by decreasing angioten-

sin-converting enzyme activity in several tissues [100–102]

and it also decreased Ang II levels in adrenal cortex

[103, 104], pituitary gland [103, 105], and kidney [106].

Heart Fail Rev (2008) 13:355–365 359

123

Furthermore, estrogen also reduces tissue responsiveness to

Ang II [106, 107]. Although some reports indicate that

estrogen activates angiotensinogen [108–110] and increa-

ses circulating levels of angiotensin [111], the overall

effects of the hormone is to limit the production of the

vasoconstrictor Ang II and to downregulate the estrogen of

the RAS may protect against the development of hyper-

tension [112]. The estrogen loss can amplify the activity of

the RAS by increasing AT1 density in Ang II target tissues

including adrenal glands. In fact, some authors have

observed significantly higher AT1 receptors in adrenal

cortex of ovariectomized rats [112]. In human, other

studies have also shown that plasma renin activity is higher

in postmenopausal women than in premenopausal women

or postmenopausal women receiving hormone replacement

therapy [113]. With these data we conclude that the

changes described by other authors in the active peptides of

RAS in the different levels of HPA axis in presence/

absence of estrogen may be due to the alterations of the

enzymes involved in their metabolism. Furthermore, it

seems that it is neither the levels of estradiol nor proges-

terone but a delicate balance between them, is responsible

for the effects on RAS-regulating aminopeptidase activities

and AVP-DA in the different levels of HPA axis and

therefore on their peptide hormone substrate.

Steroidogenesis

Finally, and in view of the precedent results, we took a step

forward and analyzed the role of RAS on steroidogenic

function to really understand the origin of the relationship

between sexual hormones and hypertension.

It was known that RAS has been found in the female

reproductive system in several mammals. Thus, binding

sites for Ang II have been identified in rat ovarian follicles

[114, 115] and corpora lutea [116, 117]. Renin- and pro-

renin-like activities have been shown in bovine follicles

[118] and ACE is also present in rat ovarian follicles and

corpora lutea [119].

Furthermore, evidence has been accumulating that Ang

II locally regulates ovulation [120, 121], oocyte maturation

[120, 121], and steroidogenesis [122]. These findings

suggest that the ovarian RAS may function independently

or in concert with the systemic RAS and may regulate

ovarian function through the paracrine and autocrine

actions of Ang II.

In the same way, several studies have provided evidence

for the presence of the RAS in testes. Immunoreactive

renin has been detected in Leydig cell of rat and human

testes [123, 124]. Similarly, other studies have shown

the presence of renin, Ang I, and Ang II in normal rat

Leydig cells and a murine Leydig cell line [125, 126].

Furthermore, ACE activity was demonstrated in rat testis

and shown to be localized predominantly in the germinal

cells, whereas only minor activity was found in the purified

adult rat Leydig and Sertoli cells [127, 128], where the

existence of a new ACE homologue (ACE2) has been

recently described [129]. Also, angiotensin receptors have

been described. In any case, it has been postulated a

putative role of Ang II in modulating the action of gona-

dotropin in Leydig cells, and therefore, on steroidogenesis

[130], inhibiting testosterone production in Leydig cells

[130].

Noradrenaline has been reported to modulate ovarian

steroidogenesis [131–137] instead of stimulating ovulation.

These effects of noradrenaline probably are mediated by

both a- and b-adrenergic receptors located in the ovary

[131–134, 137]. However, whereas it has been described

that phenylonephrine, and a1-adrenergic receptor agonist,

stimulates P secretion in cultured granulosa cells obtained

from mature rats [137]; noradrenaline has been found to

inhibit chorionic gonadotropin-induced secretion of P in

cultured human granulosa-lutein cells, most likely acting

via a-adrenergic receptors [136].

In the same way, several in vivo and in vitro systems

provided direct or indirect evidence for the ability of

testicular inervation and catecholamines (mainly nor-

adrenaline) to influence steroid production in the testis.

These effects of catecholamines on testicular testosterone

production seems to be mediated by a1-adrenergic recep-

tors [138, 139]. Furthermore, testicular RAS has also been

implicated in testicular steroidogenesis, inhibiting testos-

terone production [130]. Therefore, the precise functional

involvement of noradrenaline on steroidogenesis remains to

be established.

With these data, we analyzed the relationship between

ovarian and testicular RAS, through their proteolytic reg-

ulatory enzymes and a1-adrenergic receptors, by using their

long-acting selective antagonist doxazosin, to clarify their

roles in ovarian and testicular steroidogenic function

through the analysis of serum levels of E2 and P and

testosterone.

In our study of RAS cascade with female rats treated with

doxazosin, we have found an increase of ovarian APA

induced by the blockade of a1-adrenergic receptors induced

by doxazosin, suggesting an increased metabolism of Ang II

to Ang III. Furthermore, we have found a decrease of both

APN and APB in doxazosin-treated rats, supporting also a

decreased metabolism of Ang III. In both cases, a decrease of

Ang II levels and an increase of Ang III levels is promoted.

Furthermore, we have also found an increase in serum

levels of P after doxazosin treatment, whereas E2 levels did

not change. It has been described that E2 secretion by

quartered ovaries from pregnant mare serum gonadotropin-

treated immature rats is stimulated by Ang II, whereas P

360 Heart Fail Rev (2008) 13:355–365

123

secretion is not affected by Ang II [140, 141]. Moreover,

experiments with the in vitro-perfused rabbit ovary also

demonstrated a significant stimulatory effect of Ang II on

E2 production but not on P production [121, 122]. In

addition, exposure to Ang II receptor antagonist saralasin

blocks the HCG-stimulated ovarian production of E2 in a

concentration-dependent manner [121].

The steroidogenic effect of Ang II appears to be specific

to E2, suggesting that the Ang II-induced production of this

hormone is an ovarian RAS process, probably through

autocrine or paracrine mechanism of Ang II function in rat

ovarian follicles [140]. In the same way, it has also been

demonstrated that Ang II may interfere with the process of

steroidogenesis in porcine granulosa cells in primary cul-

ture, as shown by the concentration-dependent reduction in

the gonadotropin-induced accumulation of P produced by

Ang II [142].

In males, our results demonstrated that in vivo admin-

istration of doxazosin highly decreased serum circulating

levels of testosterone. Concomitantly, an increased specific

activity of Glu- and AspAP are found in soluble (GluAP)

and in both soluble and membrane-bound (AspAP) frac-

tions of testes. Because APA is the enzyme involved in the

degradation of Ang II to Ang III, we can postulate the

existence of low levels of Ang II against higher levels of

Ang III at testicular level. However, it is well described

that Ang II inhibits both basal and gonadotropin-stimulated

testosterone production, by inhibiting adenylate cyclase

activity in Leydig cells. Therefore, an increase, but not a

decrease of testosterone could be expected. Furthermore, to

our knowledge, it has not been described in the literature

the putative role for Ang III in testicular steroidogenesis.

Taken together, our results suggested that ovarian P

production and testosterone testicular production are also

processes regulated by RAS; Ang III is the angiotensin

responsible for that production. Therefore, it may be that the

levels of neither Ang II nor Ang III are responsible for

ovarian and testicular steroidogenesis regulation, but rather

the conversion of Ang II to Ang III, similar to the case of the

mechanism that is hypothesized for the central regulation of

blood pressure by angiotensins [143]. In this hypothesis,

Ang III produced by the action of APA activity on Ang II is

the major effector peptide of the brain RAS, exerting a tonic

stimulatory effect in the control of blood pressure. How-

ever, the similar affinities of Ang II and Ang III for the AT1

receptor do not explain why the conversion of brain Ang II

to Ang III is required to increase blood pressure. Until now,

two reasons have been considered: first, there may be

another unknown, AT1 receptor and second, it is possible

that blocking the conversion to Ang II to Ang III favors the

activation of other metabolic pathways inactivating Ang II

and producing, in each case, peptide fragments unable to

bind to AT1 and AT2 [45, 143, 144].

We concluded that ovarian P production and testicular

testosterone production, at least in the rat, are regulated by

catecholamines through a mechanism of action in which

a1-adrenergic receptors and the RAS are involved, with a

putative role for Ang III. The existence of a relationship

between catecholamines and RAS in the regulation of

ovarian functions, such as steroidogenesis, but probably

also others that need to be investigated, could be extremely

useful in understanding the disturbances in ovarian physi-

ology. On the other hand, because doxazosin is usually

used as pharmacological therapy in the treatment of

hypertension and benign prostatic hyperplasia, our results

could also indicate that the benefits are due, at least in part,

by decreasing serum circulating levels of testosterone.

With these studies we can conclude that the gender

differences observed in blood pressure may be due to the

influence of sexual hormones on the enzymes involved in

the metabolism of the active peptides of systemic and tis-

sular RAS; therefore, angiotensinases may be the key for

the knowledge of these differences.

Acknowledgement Supported by Junta de Andalucıa through Plan

Andaluz de Investigacion PAI CVI-296.

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