Francisco Hermoso

Review: Brain Aminopeptidases and Hypertension

The brain aminopeptidases that participate in the enzymatic cascade of the renin-angiotensin system play a major role in blood pressure (BP) control, and their study offers new perspectives for the understanding of central BP control and the treatment of hypertension. In this system, angiotensin II is converted to angiotensin III (Ang III) by glutamyl aminopeptidase (GluAP) and Ang III is further metabolised to angiotensin IV by alanyl aminopeptidase or arginine-aminopeptidase. It is now clear that Ang III is the key active form of the central angiotensins, exerting tonic stimulatory control over BP Therefore, the development of GluAP inhibitors as potential antihypertensive agents offers new perspectives for therapy. Brain aspartyl JR aminopeptidase, which converts angiotensin I to angiotensin 2-10, is also a possible target for antihypertensive therapy because of its potential role in BP control. Finally, since changes in BP levels, that paralleled changes in brain and plasma aminopeptidase activities, were observed after unilateral lesions of the nigrostriatal system, brain asymmetry, aminopeptidase activities and BP control appear to be related, resulting their interplay in an asymmetrical neuroendocrine response that differentially affect BP control.The study of this interaction may contribute to our understanding of how the brain controls BP

Sex differences and age-related changes in human serum aminopeptidase A activity

Given that aminopeptidase A is primarily responsible for cleaving aspartic acid and converting angiotensin II to angiotensin III, the purpose of the present study was to evaluate the activity of aminopeptidase A by determination of glutamate aminopeptidase activity (GluAP) and aspartate aminopeptidase activity (AspAP) (reported respectively as aminopeptidase A and angiotensinase A activities) in human serum during development and ageing, in an apparently healthy population of 139 male and 148 female subjects. To measure GluAP and AspAP we used glutamyl- and aspartyl-2-naphthylamide as substrates. Significant age-related increases were observed in GluAP activity in males and females and in AspAP activity in females. In males, there were no age-related differences in AspAP activity. A significant correlation was observed between age and GluAP activity in the population analysed as a whole or according to sex. No correlation was demonstrated between age and AspAP activity either in the whole population or according to sex. These results may reflect the evolution of the functional status of susceptible circulating substrates during development and ageing.

Asymmetrical response of aminopeptidase A and nitric oxide in plasma of normotensive and hypertensive rats with experimental hemiparkinsonism.

Aminopeptidases and dopamine (DA) exhibit asymmetries in the brain that are reflected in the peripheral response to unilateral striatal DA depletions (experimental hemiparkinsonism). This might be due to asymmetries in the autonomic innervation of the peripheral vessels. Nitric oxide (NO) is released through vascular sympathetic activation. A similar pathway could be postulated for aminopeptidases. Angiotensin II, metabolized by aminopeptidase A (AP A), interacts with NO and dopamine in the control of blood pressure. Moreover, plasma AP A activity and NO concentrations are elevated in hypertensive rats in which sympathetic activity is increased. We hypothesize that plasma AP A activity and NO concentrations may reflect a central asymmetry of the sympathetic activity. Therefore, we analyzed the effect of unilateral depletions of brain DA by injecting 6-hydroxydopamine into the left or right striatum and measuring plasma AP A, NO and systolic blood pressure (SBP) in normotensive and hypertensive rats. Changes in plasma AP A and NO in opposite directions may reflect an asymmetry in the function of the nigrostriatal system. Our results also revealed an inverse correlation between AP A and NO, in normotensive rats lesioned or sham operated in the right side and hypertensive rats lesioned in the left one. We concluded that the observed changes in plasma NO and AP A after left or right striatal DA depletions may be due to asymmetries in the peripheral autonomic innervation of the vessels.

Angiotensinase activities in the kidney of renovascular hypertensive rats

In spite of the well-known contribution of angiotensin II (Ang II) in the pathogenesis of Goldblatt two-kidney one clip (G2K1C) hypertension, the importance of other Ang peptides, such as Ang III, Ang IV or Ang 2-10, is scarcely understood. The functional status of these peptides depends on the action of several aminopeptidases called angiotensinases. The metabolism of Ang III to Ang IV by aminopeptidase M (AlaAP) and of Ang I to Ang 2-10 by aspartyl aminopeptidase (AspAP) was evaluated in the renal cortex and medulla of normotensive (Sham-operated) and hypertensive (G2K1C) rats, treated or not with the AT(1) receptor antagonist valsartan. The results demonstrated a highly significant increase of membrane-bound (MEMB) AlaAP in the cortex of the non-ischemic kidney of G2K1C rats compared with the kidney of normal rats and with the clipped kidney of G2K1C rats. This suggests an increased formation of Ang IV in the non-clipped kidney of G2R1C rats. Valsartan reduced MEMB AlaAP and AspAP activities in the renal cortex of normotensive and in the clipped kidney of hypertensive rats. The reduced metabolism of Ang III may prolong its half-life in valsartan-treated animals. These results suggest a role for AlaAP in renovascular hypertension. In addition, the higher AspAP activity of the renal cortex compared to medulla reflects its relative functional difference between both locations.

Oral administration of losartan influences aminopeptidase activity in the frontal cortex

Although there is a brain renin-angiotensin system, its mechanisms of control are not fully understood. We studied the effect of oral administration of the AT(1) receptor antagonist losartan on brain aminopeptidase (AP) activity, which plays a major role in neuropeptide metabolism. Six AP activities, related and non-related with the angiotensin (Ang) metabolism, were measured in their soluble and membrane-bound forms in the frontal cortex of control animals and rats treated with losartan, chronically administered via the drinking water. The results demonstrate that soluble pGluAP and membrane-bound AspAP and GluAP increased significantly in losartan-treated animals, indicating that the blockade of the AT(1) receptor stimulates the activity of AP involved in the Ang metabolism. Moreover, the blockade of the AT(1) receptor induces changes not only in the brain angiotensin metabolism, but probably also in that of other neuropeptides.

Atrial angiotensinase activity in hypothyroid, euthyroid, and hyperthyroid rats.

Abstract: Thyroid dysfunction produces marked cardiovascular responses. Hypothyroidism and hyperthyroidism cause important changes in the circulating renin-angiotensin system (RAS). Modifications in cardiac RAS have also been involved in cardiovascular alterations. Studies have revealed that thyroid hormones activate some components of cardiac RAS. Angiotensin (Ang) peptides are regulated by the activity of several aminopeptidases (AP) called angiotensinases. Previous results in our laboratory have demonstrated that thyroid dysfunction altered angiotensinase activities in hypothalamus, pituitary, and kidney. In the present study, we investigated the relationship between thyroid status and local angiotensinase activities in the atrium of hypothyroid, euthyroid, and hyperthyroid adult male rats. We have determined fluorometrically soluble and membrane-bound alanyl, glutamyl, and aspartyl aminopeptidase activities using naphthylamide derivatives as substrates. These activities have been, respectively, involved in the metabolism of Ang III to Ang IV, Ang II to Ang III, and Ang I to des-Asp Ang I. Hyperthyroidism was induced with subcutaneous injections of tetraiodothyronine (300 μg/kg/day), and the hypothyroid rats were obtained with 0.03% methimazole via the drinking water. Compared with that in euthyroid rats, a highly significant increase (by 50%) of soluble aspartyl aminopeptidase activity (P < 0.001) was observed in the atrium of hyperthyroid and hypothyroid animals. In membrane fractions, T4 treatment produced an increase in alanyl aminopeptidase (37%; P < 0.05) and aspartyl aminopeptidase activities (30%; P < 0.01). These results suggest higher formation of des-Asp Ang I in both hypothyroid and hyperthyroid rats but also suggest higher metabolism of Ang III to Ang IV in hyperthyroid animals, which is in agreement with the described alterations of cardiac RAS after thyroid dysfunction.

Papel de las aminopeptidasas en el control neuroendocrino de la presión arterial en animales de experimentación

En el control de la presion arterial participan varias enzimas proteoliticas –incluidas en el llamado sistema renina-angiotensina– que producen diversos peptidos activos que son los agentes efectivos del sistema. El estudio de estas enzimas resulta esencial para conocer en profundidad el mecanismo de control de la presion arterial y puede ofrecer la posibilidad de controlar dicho sistema con farmacos. Una glutamato aminopeptidasa transforma la angiotensina II en angiotensina III. Esta a su vez es transformada en angiotensina IV por la alanina o arginina aminopeptidasa. La angiotensina I, por accion de la aspartato aminopeptidasa, se transforma en angiotensina 2-10, a la que se han atribuido acciones contrapuestas a las hipertensivas de la angiotensina II. La angiotensina III es la forma mas activa de las angiotensinas cerebrales y tiene un efecto estimulador tonico de la presion arterial. El estudio de la inhibicion de la glutamato aminopeptidasa, por lo tanto, ha permitido el desarrollo de agentes que actuan eficazmente reduciendo la presion arterial. Asimismo, el desarrollo de activadores de la aspartato aminopeptidasa constituye otro posible objetivo para el diseno de nuevos agentes antihipertensivos. Nuestro grupo de investigacion ha observado que las lesiones unilaterales del sistema nigroestriatal en ratas da lugar a modificaciones simultaneas de la presion arterial y de la actividad aminopeptidasica cerebral y plasmatica, curiosamente dependiente del lado de la lesion. Esta posible interaccion entre presion arterial, actividad aminopeptidasica y asimetria cerebral, que daria lugar a una respuesta neuroendocrina diferenciada sobre el control de la presion arterial, podria ayudarnos a comprender el mecanismo intimo por el cual el cerebro controla en la circulacion la presion arterial.Control of blood pressure is partially accomplished by several proteolytic enzymes included in the renin-angiotensin system. These enzymes produce several peptides that form the active components of the system. Study of these enzymes is essential for a deep understanding of blood pressure control and could offer the possibility of controlling this system pharmacologically. Glutamylaminopeptidase converts angiotensin II into angiotensin III, which in turn is converted into angiotensin IV by an alanyl or arginyl aminopeptidase. Angiotensin I, through the action of aspartyl aminopeptidase, is converted into angiotensin 2-10, which may counteract the hypertensive actions of angiotensin II. Angiotensin III is the most active form of brain angiotensins and has a tonic stimulatory effect on blood pressure. Analysis of glutamyl-aminopeptidase inhibition has allowed the development of agents that effectively reduce blood pressure. Moreover, the development of aspartyl-aminopeptidase activators could be another goal, with a view to designing new antihypertensive agents. Our group has observed that unilateral lesions of the nigrostriatal pathway in rat brain produce simultaneous modifications in blood pressure and aminopeptidase activities, both in brain and plasma, curiously depending on the side of the lesion. This possible interaction among blood pressure, aminopeptidase activities and brain asymmetry, which could produce a differentiated neuroendocrine response on blood pressure control, may help us to understand the deep mechanism by which the brain is able to control blood pressure peripherally.

Reduction of cell density dependent antigenic properties of intracellular Ca2+‐regulating membranes by isoflurane in human endothelial cells

An established cell line of human umbilical vein endothelial cells displays a pronounced shift in the distribution of intracellular Ca2+‐regulating membranes as the cells grow towards confluence. In sparsely populated cultures a linearly oriented punctate pattern of vesicle‐like structures is observed similar to the distribution found in many other eucaryotic cells. As the cell population increases, the membranes condense around the nucleus. In completely confluent cultures when the cells cease to proliferate, the antigen is no longer detectable by immunofluorescence; its absence is confirmed by Western blotting experiments. Double‐labeling with antitubulin or phalloidin shows that the distribution of microtubules is not related to the distribution of the Ca2+‐regulating membranes whereas the actin fibers are superimposable onto the linearly oriented punctate vesicle‐like structures. If proliferating cells are treated with the volatile anesthetic isoflurane, the Ca2+‐regulating membranes can no longer be detected with the antibody; however, in Western blots the antigen is still present. Staining is restored after the removal of isoflurane. Such a temporary disappearance of the immunocytochemical staining might be explained by a transient oxidation and subsequent configuration change of the antigen, rendering the epitope inaccessible to the antibody, since a treatment with low concentrations of NaBH4 of cells exposed to isoflurane restores the access of the antigen to the antibody.