Ana Belén Villarejo

The Renin-Angiotensin System: New Insight into Old Therapies

Although the renin-angiotensin system (RAS) is already an old acquaintance, there are often exciting discoveries that improve our knowledge of it and open new therapeutic possibilities. Moreover, well-established drugs, such as angiotensin-converting enzyme inhibitors (ACEI), angiotensin receptor blockers (ARB), or beta-blockers, show that their mechanism of action may be the result of parallel pathways other than the ones initially established. A detailed analysis of the RAS can be carried out in part through the study of the enzymes, named angiotensinases, involved in its cascade, whose activity is a reflection of the functionality of their peptide substrates. The study of these enzymes offers the possibility of controlling the effects of angiotensins through various pharmacological manipulations. For example, angiotensinase inhibitors or activators are being used or have been proposed as antihypertensive agents. They have also been suggested as analgesic and antidepressant drugs or targets for drug development against different pathologies such as Alzheimers disease, epilepsy or ischemia. On the other hand, the analysis of brain asymmetry has revealed surprising results about the laterality of central and peripheral components of the RAS. Such studies indicate that the neurovisceral integration, already proposed by Claude Bernard (1867) should also be analyzed from a bilateral perspective. In this review, the RAS and the role of various angiotensinases implicated in the cascade are revisited. Therapeutic strategies involving some components of the RAS with an unusual vision resulting from a bilateral perspective added to their study are discussed.

Salt sensitivity in experimental thyroid disorders in rats

This study assessed salt sensitivity, analyzing the effects of an increased saline intake on hemodynamic, morphological, and oxidative stress and renal variables in experimental thyroid disorders. Six groups of male Wistar rats were used: control, hypothyroid, hyperthyroid, and the same groups treated with salt (8% via food intake). Body weight, blood pressure (BP), and heart rate (HR) were recorded weekly for 6 wk. Finally, BP and HR were recorded directly, and morphological, metabolic, plasma, and renal variables were measured. High-salt intake increased BP in thyroxine-treated rats but not in control or hypothyroid rats. High-salt intake increased cardiac mass in all groups, with a greater increase in hyperthyroid rats. Urinary isoprostanes and H(2)O(2) were higher in hyperthyroid rats and were augmented by high-salt intake in all groups, especially in hyperthyroid rats. High-salt intake reduced plasma thyroid hormone levels in hyperthyroid rats. Proteinuria was increased in hyperthyroid rats and aggravated by high-salt intake. Urinary levels of aminopeptidases (glutamyl-, alanyl-, aspartyl-, and cystinylaminopeptidase) were increased in hyperthyroid rats. All aminopeptidases were increased by salt intake in hyperthyroid rats but not in hypothyroid rats. In summary, hyperthyroid rats have enhanced salt sensitivity, and high-salt intake produces increased BP, cardiac hypertrophy, oxidative stress, and signs of renal injury. In contrast, hypothyroid rats are resistant to salt-induced BP elevation and renal injury signs. Urinary aminopeptidases are suitable biomarkers of renal injury.

Brain, Heart and Kidney Correlate for the Control of Blood Pressure and Water Balance: Role of Angiotensinases

The renin-angiotensin system (RAS) plays a major role in the control of blood pressure (BP) and water balance by coordinating brain, heart and kidney functions, connected with each other by hormonal and neural mechanisms through the autonomic nervous system (ANS). RAS function may be monitored by the study of the enzymes (angiotensinases) involved in the metabolism of its active peptides. In order to study the relationship between the brain-heart-kidney axis and the control of BP and water balance, we analyzed the correlation of angiotensinase activities, assayed as arylamidase activities, between hypothalamus, left ventricle, renal cortex and renal medulla, collected from Wistar-Kyoto and spontaneously hypertensive rats, treated or not treated with L-NAME [N(G)-nitro-L-arginine methyl ester]. This compound not only inhibits the formation of nitric oxide but also disrupts the normal function of the ANS activating the sympathetic nervous system (SNS) to increase BP. In addition, to assess the influence of the SNS, we studied the effect of its blockade by treatment of both strains with propranolol. The present results support the notion that RAS function of the brain-heart-kidney axis, as reflected by the activities of angiotensinases, is reciprocally connected by afferent and efferent mechanisms between these locations, presumably through the ANS. These results reveal new aspects of neuroendocrine regulation possibly involving the ANS.

Angiotensinase and vasopressinase activities in hypothalamus, plasma, and kidney after inhibition of angiotensin-converting enzyme: basis for a new working hypothesis.

Reducing angiotensin II (Ang II) production via angiotensin-converting enzyme (ACE) inhibitors is a key approach for the treatment of hypertension. However, these inhibitors may also affect other enzymes, such as angiotensinases and vasopressinase, responsible for the metabolism of other peptides also involved in blood pressure control, such as Ang 2-10, Ang III, Ang IV, and vasopressin. We analyzed the activity of these enzymes in the hypothalamus, plasma, and kidney of normotensive adult male rats after inhibition of ACE with captopril. Aspartyl- (AspAP), glutamyl- (GluAP), alanyl- (AlaAP) and cystinyl-aminopeptidase (CysAP) activities were measured fluorimetrically using arylamides as substrates. Systolic blood pressure (SBP), water intake, and urine flow were also measured. Captopril reduced SBP and increased urine flow. In the hypothalamus, GluAP and AspAP increased, without significant changes in either AlaAP or CysAP. In contrast with effects in plasma, GluAP was unaffected, AspAP decreased, while AlaAP and CysAP increased. In the kidney, enzymatic activities did not change in the cortex, but decreased in the medulla. These data suggest that after ACE inhibition, the metabolism of Ang I in hypothalamus may lead mainly to Ang 2-10 formation. In plasma, the results suggest an increased formation of Ang IV together with increased vasopressinase activity. In the kidney, there is a reduction of vasopressinase activity in the medulla, suggesting a functional reduction of vasopressin in this location. The present data suggest the existence of alternative pathways in addition to ACE inhibition that might be involved in reducing BP after captopril treatment.

Tissue distribution of CysAP activity and its relationship to blood pressure and water balance

AIMS To better understand the functional role of soluble (Sol) and membrane-bound (MB) cystinyl-aminopeptidase (CysAP) activities, we studied differentially their organ distribution in adult male Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR)with or without treatment with captopril.We searched for a possible tissue-specific association of CysAP with water balance and blood pressure. MAIN METHODS We used twenty WKY rats distributed in ten controls and ten captopril-treated, and sixteen SHR divided in eight controls and eight captopril-treated. Captopril (100 mg/kg/day) was administered in drinking water for 4 weeks. Systolic blood pressure, water intake and diuresis were measured individually. CysAP was assayed fluorometrically using L-cystine-di-β-naphthylamide as substrate. KEY FINDINGS Sol or MB activities were generally higher in SHR compared to WKY notably in hypothalamus and kidney than in the other tissues. Captopril mainly decreased CysAP in SHR whereas it increased in WKY. The distribution of Sol CysAP was more homogeneous among tissues ofWKY than SHR. In contrast, the distribution of MB CysAP was more heterogeneous than Sol CysAP in both WKY and SHR. This suggests that MB CysAP activity acts in a more tissue-specific manner than Sol CysAP. The majority of the significant correlations between tissue activities and the measured physiological parameters were observed mostly in renal medulla and hypothalamus. SIGNIFICANCE Sol and MB CysAP activities, acting separately or in concert and mainly in renal medulla, regulate the function of their susceptible endogenous substrates, and may participate meaningfully in the control of blood pressure and fluid balance.

Interaction of neuropeptidase activities in cortico-limbic regions after acute restraint stress

Brain enkephalin, vasopressin and oxytocin are anxiolytic agents involved in the stress response. Acute restraint stress influences certain neuropeptidase activities, such as some enkephalin-degrading peptidases and vasopressinase/oxytocinase, in the medial prefrontal cortex (mPFC), amygdala (AM) or hippocampus (HC), which are involved in this response. Because these regions form a unified circuit and cooperate in their response to stress, it is important to analyze the profile of the regional distribution of these activities as well as their inter-regional model of interaction in this circuit. Regarding the regional study, although most activities showed a marked predominance of the AM over the HC and mPFC, both in control and stressed animals, enkephalin-degrading activity, assayed as membrane-bound alanyl aminopeptidase activity, showed a change after stress, increasing in the HC and decreasing in the AM. The correlational study in controls indicated essentially a positive interaction between the mPFC and AM. In marked contrast, there was a highly significant change in the functional status of this circuit after stress, showing mainly a positive correlation between the mPFC and HC and between the AM and HC. The existence of correlations does not demonstrate a direct relationship between regions. However, reasons for such strong associations after restraint stress should be examined. The present study may indicate a connection between neuropeptidase activities and their corresponding neuropeptidergic substrates due to significant changes in the functional status of the cortico-limbic circuit after restraint stress.

The Brain-Heart Connection: Frontal Cortex and Left Ventricle Angiotensinase Activities in Control and Captopril-Treated Hypertensive Rats—A Bilateral Study

The model of neurovisceral integration suggests that the frontal cortex (FC) and the cardiovascular function are reciprocally and asymmetrically connected. We analyzed several angiotensinase activities in the heart left ventricle (VT) of control and captopril-treated SHR, and we search for a relationship between these activities and those determined in the left and right FC. Captopril was administered in drinking water for 4 weeks. Samples from the left VT and from the left and right FC were obtained. Soluble and membrane-bound enzymatic activities were measured fluorometrically using arylamides as substrates. The weight of heart significantly decreased after treatment with captopril, mainly, due to the reduction of the left VT weight. In the VT, no differences for soluble activities were observed between control and treated SHR. In contrast, a generalized significant reduction was observed for membrane-bound activities. The most significant correlations between FC and VT were observed in the right FC of the captopril-treated group. The other correlations, right FC versus VT and left FC versus VT in controls and left FC versus VT in the captopril group, were few and low. These results confirm that the connection between FC and cardiovascular system is asymmetrically organized.

Influence of extra virgin olive oil on blood pressure and kidney angiotensinase activities in spontaneously hypertensive rats.

High-fat diets are associated with the development of cardiovascular diseases. The efficacy of the current strategies of treatment is still not entirely satisfactory, and new approaches are being considered. To analyze the beneficial effects of extra virgin olive oil as a major component of the Mediterranean diet, we studied systolic blood pressure and angiotensinase activities, since this enzyme is involved in the metabolism of angiotensins, in the kidney of hypertensive rats fed during 12 weeks with a diet enriched with extra virgin olive oil compared with a standard diet. As a reflex of oxidative stress, 8-isoprostanes and nitric oxide were quantified in urine. Results demonstrated a progressive increase in systolic blood pressure until the end of the feeding period in both groups. However, this increase was delayed in the extra virgin olive oil group until week six, with the systolic blood pressure being always lower in this group. Nitric oxide and 8-isoprostanes were lower in the extra virgin olive oil group. While we can deduce a higher formation of angiotensin 2-10 in the renal cortex, a higher availability of angiotensin II may be presumed in the renal medulla of animals fed an extra virgin olive oil diet than in animals fed a standard diet. Our results support the beneficial influence of extra virgin olive oil on cardiovascular function and suggest that the Mediterranean diet may be beneficial in itself but it may also be an effective tool in the treatment of hypertension.

Asymmetrical effect of captopril on the angiotensinase activity in frontal cortex and plasma of the spontaneously hypertensive rats: expanding the model of neuroendocrine integration.

There is a reciprocal connection between the frontal cortex (FC) and cardiovascular function, and this connection is functionally lateralized. The possible pathophysiological impact of neuroendocrine asymmetries is largely underestimated. Our aim was to examine the activity of soluble (SOL) and membrane-bound (MB) aminopeptidases (APs) involved in the renin-angiotensin system in the peripheral plasma and in the left and right FC, in both untreated (control) and captopril-treated spontaneously hypertensive rats (SHRs). Enzymatic activities were measured fluorometrically using arylamide derivatives as substrates. Captopril reduced systolic blood pressure, but no differences in plasma AP activity were observed between the control and treated SHRs. In contrast, whereas the bilateral pattern (left vs. right differences) of SOL activities did not substantially change in the FC after captopril treatment, the asymmetries observed for MB activities in the FC markedly increased compared with the control group. Moreover, correlations between the AP activities in the plasma and those in the left or right FC were observed. In the control rats, the plasma AP activities correlated significantly with those in the right FC, whereas they correlated with those in the left FC in the captopril-treated group. In both groups (control and captopril), these correlations were negative for the SOL activity but positive for the MB activity. The present results reveal a pattern of bilateral behavior between the nervous and cardiovascular systems. The inverted bilateral behavior after captopril treatment suggests a systematized, lateralized neuroendocrine response representing a regular bilateral behavior that has yet to be analyzed.

Effects of antihypertensive drugs on angiotensinase activities in the testis of spontaneously hypertensive rats.

Sexual dysfunction is a frequent adverse effect during antihypertensive therapy. However, the mechanisms responsible for these effects are not well understood. The renin-angiotensin system has been identified in testis where it may play a role in testicular function and be involved in the detrimental effects of antihypertensive drugs. Therefore, our objective was to compare the influence of captopril and propranolol on plasma testosterone levels and on hydrolyzing angiotensins enzymes (angiotensinases) in the testis of spontaneously hypertensive rats (SHRs) and in control animals. Twenty-four adult male SHRs were used in this study; eight were treated with captopril in drinking water, 8 with propranolol, and 8 were controls. At the end of the 4 weeks treatment period, systolic blood pressure (SBP) was recorded, blood samples were collected, and the right testis was dissected after perfusion of the rat with saline. The soluble (Sol) and membrane-bound (MB) fractions were obtained after solubilization and ultracentrifugation. Fluorometric measurement of Sol and MB angiotensinase activities were performed using arylamide derivatives as substrates. Testosterone was measured by enzyme immunoassay. SBP decreased after captopril but did not change with propranolol treatment. Whereas captopril did not affect angiotensinase activities, highly significant reductions in Sol and MB angiotensinase activities, particularly glutamyl- and aspartyl-aminopeptidases, were observed after treatment with propranolol. Plasma testosterone decreased in captopril treated rats but propranolol had a greater effect. The present results support a general functional depression of the RAS cascade in the testis of propranolol-treated SHR, which may influence the sexual function of these animals.