María Jesús García

Hormonal status modifies renin-angiotensin system-regulating aminopeptidases and vasopressin-degrading activity in the hypothalamus-pituitary-adrenal axis of male mice

Local renin-angiotensin systems (RAS) have been postulated in brain, pituitary and adrenal glands. These local RAS have been implicated, respectively, in the central regulation of the cardiovascular system and body water balance, the secretion of pituitary hormones and the secretion of aldosterone by adrenal glands. By other hand, it is known that the hypothalamus-pituitary-adrenal (HPA) axis is involved in blood pressure regulation, and is affected by sex hormones. The aim of the present work is to analyze the influence of testosterone on RAS-regulating aminopeptidase A, B and M activities and vasopressin-degrading activity in the HPA axis, measuring these activities in their soluble and membrane-bound forms in the hypothalamus, pituitary and adrenal glands of orchidectomized males and orchidectomized males treated subcutaneously with several doses of testosterone. The present data suggest that in male mice, testosterone influences the RAS- and vasopressin-degrading activities at all levels of the HPA axis.

Diet-induced hypercholesterolemia impaired testicular steroidogenesis in mice through the renin-angiotensin system

Hypercholesterolemia and low testosterone concentrations in men are associated with a high risk factor for atherosclerosis. It is known that cholesterol serves as the major precursor for the synthesis of the sex hormones. The bioactive peptides of the renin-angiotensin-system localized in the gonads play a key role in the relation between cholesterol and testosterone by modulating steroidogenesis and inhibiting testosterone production. In the present work, we evaluated the effects of diet-induced hypercholesterolemia on circulating testosterone levels and its relationship with the testicular RAS-regulating specific aminopeptidase activities in male mouse. A significant decrease in serum circulating levels of testosterone was observed after induced hypercholesterolemia. The changes found in aminopeptidase activities suggest a role of Ang III and Ang IV in the regulation of steroidogenesis.

Renin angiotensin system-regulating aminopeptidase activities in serum of pre- and postmenopausal women with breast cancer.

Angiotensin peptides regulate vascular tone and natriohydric balance through the renin angiotensin system (RAS) and are related with the angiogenesis which plays an important role in the metastatic pathway. Estrogen influences the aminopeptidases (APs) involved in the metabolism of bioactive peptides of RAS through several pathways. We analyze RAS-regulating AP activities in serum of pre- and postmenopausal women with breast cancer to evaluate the putative value of these activities as biological markers of the development of breast cancer. We observed an increase in aminopeptidase N (APN) and aminopeptidase B (APB) activities in women with breast cancer; however, a decrease in aspartyl-aminopeptidase (AspAP) activity in premenopausal women. These results suggest a slow metabolism of angiotensin II (Ang II) to angiotensin III (Ang III) in premenopausal women and a rapid metabolism of Ang III to angiotensin IV (Ang IV) in pre- and postmenopausal women with breast cancer. An imbalance in the signals activated by Ang II may produce abnormal vascular growth with different response between pre- and postmenopausal women depending on the hormonal profile and the development of the disease.

Ethanol modifies differently aspartyl- and glutamyl-aminopeptidase activities in mouse frontal cortex synaptosomes

Aminopeptidase A activity (aspartyl aminopeptidase (AspAP) and glutamyl aminopeptidase (GluAP) exerts angiotensinase activity due to its relation to the metabolism of angiotensins in the regional brain renin-angiotensin system (RAS). This activity may also modify the free amino acid pool through the release of N-terminal acidic amino acids. Ethanol (EtOH) exerts profound effects on the brain, inducing important neurological damages. Our purpose is to study the influence of EtOH on AspAP and GluAP activities on basal and K(+)-stimulated conditions, at the synapse level. We used mouse frontal cortex synaptosomes and their incubation supernatant in a Ca(2+)-containing or Ca(2+)-free artificial cerebrospinal fluid. We evaluate the possible contribution of these enzymatic activities on brain blood pressure regulation through RAS and/or the free acidic amino acid pool. The results obtained are correlated with several parameters of oxidative stress, such as free radical generation, lipid peroxidation, and protein oxidation. Under basal conditions, in synaptosomes, EtOH inhibits AspAP and GluAP activities independently of Ca(2+). In the supernatant, however, EtOH differently modulates the two enzyme activities under the various concentrations. Under K(+)-stimulated conditions, EtOH inhibits the K(+)-stimulated increase on AspAP and GluAP differently depending on the presence or absence of Ca(2+) and the concentration of EtOH used. These results invalidate the idea that excess free acidic amino acids could be released by AspAP and GluAP to induce neurodegeneration. The changes in AspAP and GluAP activities as a consequence of EtOH administration and their role in the brain RAS are discussed.

Oleate, linoleate and cholesterol differently modify aspartyl- and glutamyl-aminopeptidase activities in primary cultures of rat astrocytes.

The intake of mono- and polyunsaturated fatty acids has been associated with a minor risk of cardiovascular diseases including hypertension. Changes in levels of fatty acids may also modify the cell activity and may be related with alterations in different regulatory processes. Aminopeptidases are zinc-metalloenzymes which metabolise circulating peptide hormones in several tissues. Glutamyl-aminopeptidase (GluAP) and to a lesser extent, aspartyl-aminopeptidase (AspAP), are related with angiotensin metabolism in the renin-angiotensin system. The present work was designed to study the effect of a range of concentrations (1-100 microM) of oleic and linoleic acids and cholesterol present in the culture medium on the activity of GluAP and AspAP in the culture of rat cerebral cortical astrocytes taken from 21-day-old fetuses. The results showed that oleic acid inhibits, while linoleic acid stimulates the activity of AspAP. Both fatty acids inhibit the activity of GluAP. Cholesterol stimulates the activity of both enzymes. On the basis of these results, a functional link may exit between the effects of fatty acids on hypertension and the modulation of aminopeptidase activity by these compounds in rat astrocytes, as an example of target cell type in the central nervous system.

Renin-Angiotensin System-Regulating Aminopeptidase Activities Are Modified in the Pineal Gland of Rats with Breast Cancer Induced by N-Methyl-Nitrosourea

Objective: Pineal function has been considered particularly as a neuroendocrine modulator in hormone responsive tumors, like the hormone-dependent mammary tumors. The complexity of the gland function, moreover, is denoted by the presence of a local renin-angiotensin-system (RAS) that regulates melatonin biosynthesis. 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. Our aim is to know their role in the regulation of pineal RAS and breast cancer. Design: Aminopeptidase N (APN), aminopeptidase B (APB) and aminopeptidase A (aspartyl- and glutamyl-aminopeptidase, APA) activities are measured in the pineal gland of rats with breast cancer induced by N-methyl nitrosourea (NMU). Methods: Aminopeptidase activities were measured fluorimetrically using their corresponding aminoacyl-ß-naphthylamides as substrates. Results: Specific APN and APB activities in pineal gland of controls and NMU-treated rats were not modified. Aspartyl aminopeptidase activity significantly decreased in NMU-treated rats when compared with control group. On the contrary, glutamyl aminopeptidase activity did not show significant differences between groups. Conclusions: We propose that the local RAS in pineal gland is modified in rats with breast cancer induced by NMU through the inhibition of AspAP activity, which may lead to increased levels of Ang II. Ang II could be responsible of the overproduction of melatonin, supporting a mechanism to restrain the promotion and/or progression of breast cancer.

Specific enkephalin-degrading aminopeptidase activity in the HPT and HPO axes of rats with breast cancer induced by N-methyl nitrosourea

State and function of breast depend on an endocrinological balance, the upsetting of which can be a factor favorable to the development of cancer. Enkephalins (ENK) have been considered as a particular form of adaptation to defense to the organism against neoplastic processes. However, ENK may modify the endocrine functions of glands such as the ovary or the thyroid through the hypothalamus-pituitary axis, acting direct or indirectly as endocrine, paracrine or autocrine stimulatory growth factors. The present work analyses enkephalin-degrading tyrosyl aminopeptidase (EDA) activity in the hypothalamus-pituitary-thyroid (HPT) and hypothalamus-pituitary-ovary (HPO) axes in a rat model of breast cancer induced by N-methyl-nitrosourea (NMU) to state the relationship between ENK levels modification through EDA activity at different neuroendocrine levels and breast cancer. Results obtained show a decrease in EDA activity in hypothalamus, anterior and posterior pituitary, thyroid and ovary, suggesting increased levels of ENK in all these locations. These ENK may induce breast cancer cell growth and progression not only at breast level, but also acting at several neuroendocrine levels such as the HPT and HPO axes, inducing an unbalance of several other hormones, which could also facilitate the progression of cancer as an undesirable concomitant effect.

Calcium-dependent modulation by ethanol of mouse synaptosomal pyroglutamyl aminopeptidase activity under basal and K+-stimulated conditions

We studied the in vitro effects of ethanol (25, 50 and 100 mM) on pyroglutamyl aminopeptidase activity (pGluAP), which has been reported as thyrotrophin-releasing-hormone-degrading activity. pGluAP was measured in presence or absence of calcium, under basal and K(+)-stimulated conditions, in synaptosomes and their incubation supernatant, using pyroglutamyl-beta-naphthylamide as substrate. In basal conditions, in synaptosomes, pGluAP was inhibited by ethanol in a calcium-independent way. In the supernatant, the response differed depending on the concentration of ethanol. Depolarization with K(+) modified pGluAP in synaptosomes and supernatant depending on the presence or not of calcium. In synaptosomes, in absence of calcium, the activity was inhibited at the highest concentrations of ethanol. In contrast, in the supernatant, under depolarizing conditions, ethanol increases pGluAP in absence of calcium. These changes may be in part responsible of the behavioural changes associated to alcohol intake.

Mammary renin-angiotensin system-regulating aminopeptidase activities are modified in rats with breast cancer.

Angiotensin II in particular and/or the local renin–angiotensin system in general could have an important role in epithelial tissue growth and modelling; therefore, it is possible that it may be involved in breast cancer. In this sense, previous works of our group showed a predominating role of angiotensin II in tumoral tissue obtained from women with breast cancer. However, although classically angiotensin II has been considered the main effector peptide of the renin–angiotensin system cascade, several of its catabolism products such as angiotensin III and angiotensin IV also possess biological functions. These peptides are formed through the activity of several proteolytic regulatory enzymes of the aminopeptidase type, also called angiotensinases. The aim of this work was to analyse several specific angiotensinase activities involved in the renin–angiotensin system cascade in mammary tissue from control rats and from rats with mammary tumours induced by N-methyl-nitrosourea (NMU), which may reflect the functional status of their target peptides under the specific conditions brought about by the tumoural process. The results show that soluble and membrane-bound specific aspartyl aminopeptidase activities and membrane-bound glutamyl aminopeptidase activity increased in mammary tissue from NMU-treated animals and soluble aminopeptidase N and aminopeptidase B activities significantly decreased in mammary tissue from NMU-treated rats. These changes support the existence of a local mammary renin–angiotensin system and that this system and its putative functions in breast tissue could be altered by the tumour process, in which we suggest a predominant role of angiotensin III. All described data about the renin–angiotensin system in mammary tissue support the idea that it must be involved in normal breast tissue functions, and its disruption could be involved in one or more steps of the carcinogenesis process.

Differential Effects of Dietary Cholesterol on Aminopeptidase A, B and M in the Frontal Cortex of Male and Female Mice

Although hypercholesterolemia and hypertension have been extensively associated, the regulatory mechanism underlying this relationship is poorly understood. Systemic and local renin-angiotensin systems are involved in the control of blood-pressure. Angiotensin II has been considered as the main effector peptide of renin-angiotensin system. However, other peptides derived from the metabolism of angiotensin II, as angiotensins III and IV have been shown to play significant roles. The aim of this study is to analyse the effect of dietary cholesterol on the activity of the enzymes involved in the metabolism of angiotensins II and III. Soluble and membrane-bound aminopeptidase A (aspartyl- and glutamyl-aminopeptidases), B (arginyl-aminopeptidase) and M (alanyl-aminopeptidase) activities were measured in the frontal cortex of male and female mice fed a cholesterol enriched-diet (1% cholesterol; 0.5 cholic acid). Soluble and membrane-bound aminopeptidases B and M did not change in male or female cholesterol groups. Significant increases were observed in membrane-bound aspartyl- and glutamyl-aminopeptidase activities in both cholesterol groups. Soluble aspartyl- and glutamyl-aminopeptidases did not change in male cholesterol group, but significant decreases were detected in female cholesterol group. Our results may indicate that the metabolism of angiotensin II to angiotensin III by aminopeptidase A is increased, but angiotensin III metabolism by aminopeptidases B and M is not modified after cholesterol intake; so cholesterol may enhance the effects of angiotensin III, at least, at the cortical level.