Angiotensinase Activity Research Articles

The Type of Fat in the Diet Influences Regulatory Aminopeptidases of the Renin-Angiotensin System and Stress in the Hypothalamic-Pituitary-Adrenal Axis in Adult Wistar Rats.

(1) Background: Prolonged feeding with a high-fat diet (HFD) acts as a stressor by activating the functions of the hypothalamic-pituitary-adrenal gland (HPA) stress axis, accompanied of hypertension by inducing the renin-angiotensin-aldosterone system. Angiotensinases enzymes are regulatory aminopeptidases of angiotensin metabolism, which together with the dipeptidyl peptidase IV (DPP-IV), pyroglutamyl- and tyrosyl-aminopeptidase (pGluAP, TyrAP), participate in cognitive, stress, metabolic and cardiovascular functions. These functions appear to be modulated by the type of fat used in the diet. (2) Methods: To analyze a possible coordinated response of aminopeptidases, their activities were simultaneously determined in the hypothalamus, adenohypophysis and adrenal gland of adult male rats fed diets enriched with monounsaturated (standard diet (S diet) supplemented with 20% virgin olive oil; VOO diet) or saturated fatty acids (diet S supplemented with 20% butter and 0.1% cholesterol; Bch diet). Aminopeptidase activities were measured by fluorimetry using 2-Naphthylamine as substrates. (3) Results: the hypothalamus did not show differences in any of the experimental diets. In the pituitary, the Bch diet stimulated the renin-angiotensin system (RAS) by increasing certain angiotensinase activities (alanyl-, arginyl- and cystinyl-aminopeptidase) with respect to the S and VOO diets. DPP-IV activity was increased with the Bch diet, and TyrAP activity decrease with the VOO diet, having both a crucial role on stress and eating behavior. In the adrenal gland, both HFDs showed an increase in angiotensinase aspartyl-aminopeptidase. The interrelation of angiotensinases activities in the tissues were depending on the type of diet. In addition, correlations were shown between angiotensinases and aminopeptidases that regulate stress and eating behavior. (4) Conclusions: Taken together, these results support that the source of fat in the diet affects several peptidases activities in the HPA axis, which could be related to alterations in RAS, stress and feeding behavior.

Interaction between Angiotensinase Activities in Pituitary and Adrenal Glands of Wistar-Kyoto and Spontaneously Hypertensive Rats under Hypotensive or Hypertensive Treatments.

In the present study, we analyzed the activity of several aminopeptidases (angiotensinases) involved in the metabolism of various angiotensin peptides, in pituitary and adrenal glands of untreated Wistar–Kyoto (WKY) and spontaneously hypertensive rats (SHR) or treated with the antihypertensive drugs captopril and propranolol or with the L-Arginine hypertensive analogue L-NG-Nitroarginine Methyl Ester (L-NAME). Intra- and inter-gland correlations between angiotensinase activities were also calculated. Membrane-bound alanyl-, cystinyl-, and glutamyl-aminopeptidase activities were determined fluorometrically using aminoacyl-β-naphthylamide as substrates. Depending on the type of angiotensinase analyzed, the results reflect a complex picture showing substantial differences between glands, strains, and treatments. Alanyl-aminopeptidase responsible for the metabolism of Ang III to Ang IV appears to be the most active angiotensinase in both pituitary and adrenals of WKY and particularly in SHR. Independently of treatment, most positive correlations are observed in the pituitary gland of WKY whereas such positive correlations are predominant in adrenals of SHR. Negative inter-gland correlations were observed in control SHR and L-NAME treated WKY. Positive inter-gland correlations were observed in captopril-treated SHR and propranolol-treated WKY. These results may reflect additional mechanisms for increasing or decreasing systolic blood pressure in WKY or SHR.

The Role of High Fat Diets and Liver Peptidase Activity in the Development of Obesity and Insulin Resistance in Wistar Rats.

High-fat diets (HFD) have been widely associated with an increased risk of metabolic disorders and overweight. However, a high intake of sources that are rich in monounsaturated fatty acids has been suggested as a dietary agent that is able to positively influence energy metabolism and vascular function. The main objective of this study was to analyze the role of dietary fats on hepatic peptidases activities and metabolic disorders. Three diets: standard (S), HFD supplemented with virgin olive oil (VOO), and HFD supplemented with butter plus cholesterol (Bch), were administered over six months to male Wistar rats. Plasma and liver samples were collected for clinical biochemistry and aminopeptidase activities (AP) analysis. The expression of inducible nitric oxide synthase (iNOS) was also determined by Western blot in liver samples. The diet supplement with VOO did not induce obesity, in contrast to the Bch group. Though the VOO diet increased the time that was needed to return to the basal levels of plasma glucose, the fasting insulin/glucose ratio and HOMA2-%B index (a homeostasis model index of insulin secretion and valuation of β-cell usefulness (% β-cell secretion)) were improved. An increase of hepatic membrane-bound dipeptidyl-peptidase 4 (DPP4) activity was found only in VOO rats, even if no differences in fasting plasma glucagon-like peptide 1 (GLP-1) were obtained. Both HFDs induced changes in hepatic pyroglutamyl-AP in the soluble fraction, but only the Bch diet increased the soluble tyrosyl-AP. Angiotensinase activities that are implicated in the metabolism of angiotensin II (AngII) to AngIV increased in the VOO diet, which was in agreement with the higher activity of insulin-regulated-AP (IRAP) in this group. Otherwise, the diet that was enriched with butter increased soluble gamma-glutamyl transferase (GGT) and Leucyl-AP, iNOS expression in the liver, and plasma NO. In summary, VOO increased the hepatic activity of AP that were related to glucose metabolism (DPP4, angiotensinases, and IRAP). However, the Bch diet increased activities that are implicated in the control of food intake (Tyrosine-AP), the index of hepatic damage (Leucine-AP and GGT), and the expression of hepatic iNOS and plasma NO. Taken together, these results support that the source of fat in the diet affects several peptidases activities in the liver, which could be related to alterations in feeding behavior and glucose metabolism.

Thyroid Disorders Change the Pattern of Response of Angiotensinase Activities in the Hypothalamus-Pituitary-Adrenal Axis of Male Rats.

Thyroid disorders affect the hypothalamic-pituitary-adrenal axis with important consequences on the cardiovascular function in which the renin-angiotensin system plays a major role. Hypo and hyperthyroidism influence the classic main components of the renin-angiotensin system. However, the behavior of other elements of the renin-angiotensin system such as Ang III, Ang 2-10, Ang IV, or AT4, regulated by angiotensinase enzymes such as alanyl- (AlaAP), cystinyl- (CysAP), glutamyl- (GluAP), or aspartyl-aminopeptidase (AspAP), has not yet been described. In order to obtain a comprehensive view on the response of the renin-angiotensin system in the hypothalamic-pituitary-adrenal axis of animals with thyroid disorders, these enzyme activities were simultaneously analyzed fluorometrically, using arylamide derivatives as substrates in hypothalamus, anterior and posterior pituitary, adrenals and plasma of euthyroid, hypothyroid, and hyperthyroid rats, and their intra- and inter-tissue correlations were evaluated. The response is depending on the type of enzyme studied, its location and the thyroid status. Anterior pituitary, adrenals and plasma were mainly affected by the thyroid disorders. In the anterior pituitary, GluAP and AspAP increased in hypothyroid rats. In adrenals, AlaAP and CysAP decreased in hypothyroid whereas GluAP and AspAP decreased in hyperthyroid rats. In plasma, while AlaAP increased in hypo- and hyperthyroid rats, CysAP and GluAP decreased only in hyperthyroid. In comparison with euthyroid, intra-tissue correlations decreased in hypothyroid but inter-tissue correlations decreased mainly in hyperthyroid rats. Thyroid disorders also produced a disruption in the pattern of inter-tissue correlations observed in euthyroid. These results suggest that thyroid hormone levels hit components of the renin-angiotensin system and may influence the paracrine and endocrine cross talk between cells.

Diurnal opposite variation between angiotensinase activities in photo–neuro–endocrine tissues of rats

ABSTRACTCentral and peripheral renin–angiotensin systems (RASs) act in a coordinated manner for the physiologic functions regulated by neuroendocrine events. However, whereas the diurnal rhythm of peripheral circulatory and tissue RASs is well known, the circadian behaviour of their components in central photo–neuro–endocrine structures, key elements for the control of circadian rhythms, has been barely studied. In the present study, we analysed the aspartyl- (AspAP) and glutamyl-aminopeptidase (GluAP) (aminopeptidase A) activities, the angiotensinases responsible for the metabolism of Ang I to Ang 2–10 and Ang II to Ang III, respectively, in the retina, anterior hypothalamus and pituitary at different light and dark time-points of a 12:12 h light:dark cycle (7–19 h light), using arylamide derivatives as substrates. The results demonstrated that while retina and pituitary exhibited their highest levels of AspAP activity in the light period and the lowest in the dark one, the contrary occurred in the hypothalamus – the lowest levels were observed in light conditions and the highest in darkness. The outcome for GluAP showed the highest levels in the light period and the lowest in the dark one in the three tissues analysed. In conclusion, changes in angiotensinase activities throughout the daytime may cause changes of their respective substrates and derived peptides and, consequently, in their functions. This observation may have implications for the treatment of hypertension.

Abstract P209: RAS-Equilibrium Analysis: Simplified Biochemical Characterization of the Renin-angiotensin-system and Implications for Diagnosis and Treatment of Hypertension

The concentrations of angiotensins are maintained by equal rates of formation and degradation. The resulting steady-state angiotensin levels are affected by local molecular factors including tissue, endothelium or blood cell associated angiotensin receptors and processing enzymes as well as plasma soluble RAS components. The high concentration of the pro-hormone angiotensinogen in human plasma in combination with reported ranges for plasma renin activity result in a long-lasting and stable Ang I formation rate without significantly reducing angiotensinogen levels within several hours. This phenomenon can be utilized for the generation of an ex vivo situation that is characterized by significantly higher but stable angiotensin peptide levels. These ex vivo equilibrium angiotensin levels provide an integrated picture about plasma angiotensinase activities and therefore represent a powerful diagnostic tool for analyzing the systemic RAS in clinical samples. Moreover, these ex vivo levels remain stable over hours of incubation at 37°C and show a very high correlation (>90%) with angiotensin levels obtained by a state-of-the art sample collection procedure using complex inhibitor cocktails for stabilizing angiotensin peptides during blood collection. The quantification of equilibrium angiotensin levels does not require any special sample collection procedures and can be applied to frozen serum and heparin plasma samples. It turned out that ex vivo angiotensin levels represent a measure for the consumption of angiotensin metabolites by the organism, therefore providing a powerful and versatile tool for assessing in vivo angiotensin signaling on the patient specific level enabling new diagnosis based rationales for anti-hypertensive therapies. The application of ex vivo RAS-Fingerprinting in clinical studies could substantially enhance our understanding of the regulation and physiology of the human RAS and could further lead to the development of powerful personalized approaches in the future treatment of hypertension.

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.

Abstract 479: Ex vivo Equilibrium Analysis of the Renin-Angiotensin-System: Clinical Implications for Diagnosis and Treatment of Hypertension

The concentrations of angiotensins are maintained by equal rates of formation and degradation. The resulting steady-state angiotensin levels are affected by local molecular factors including tissue, endothelium or blood cell associated angiotensin receptors and processing enzymes as well as plasma soluble RAS components. The high concentration of the pro-hormone angiotensinogen in human plasma in combination with reported ranges for plasma renin activity result in a long-lasting and stable Ang I formation rate without significantly reducing angiotensinogen levels within several hours. This phenomenon can be utilized for the generation of an ex vivo situation that is characterized by significantly higher but stable angiotensin peptide levels. These ex vivo equilibrium angiotensin levels provide an integrated picture about plasma angiotensinase activities and therefore represent a powerful diagnostic tool for analyzing the systemic RAS in clinical samples. Moreover, these ex vivo levels remain stable over hours of incubation at 37°C and show a very high correlation (>90%) with angiotensin levels obtained by a state-of-the art sample collection procedure using complex inhibitor cocktails for stabilizing angiotensin peptides during blood collection. The quantification of equilibrium angiotensin levels does not require any special sample collection procedures and can be applied to frozen serum and heparin plasma samples. It turned out that ex vivo angiotensin levels represent a measure for the consumption of angiotensin metabolites by the organism, therefore providing a powerful and versatile tool for assessing in vivo angiotensin signaling on the patient specific level enabling new diagnosis based rationales for anti-hypertensive therapies. The application of ex vivo RAS-Fingerprinting in clinical studies could substantially enhance our understanding of the regulation and physiology of the human RAS and could further lead to the development of powerful personalized approaches in the future treatment of hypertension.

Altered glutamyl-aminopeptidase activity and expression in renal neoplasms.

BackgroundAdvances in the knowledge of renal neoplasms have demonstrated the implication of several proteases in their genesis, growth and dissemination. Glutamyl-aminopeptidase (GAP) (EC. 3.4.11.7) is a zinc metallopeptidase with angiotensinase activity highly expressed in kidney tissues and its expression and activity have been associated wtih tumour development.MethodsIn this prospective study, GAP spectrofluorometric activity and immunohistochemical expression were analysed in clear-cell (CCRCC), papillary (PRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytoma (RO). Data obtained in tumour tissue were compared with those from the surrounding uninvolved kidney tissue. In CCRCC, classic pathological parameters such as grade, stage and tumour size were stratified following GAP data and analyzed for 5-year survival.ResultsGAP activity in both the membrane-bound and soluble fractions was sharply decreased and its immunohistochemical expression showed mild staining in the four histological types of renal tumours. Soluble and membrane-bound GAP activities correlated with tumour grade and size in CCRCCs.ConclusionsThis study suggests a role for GAP in the neoplastic development of renal tumours and provides additional data for considering the activity and expression of this enzyme of interest in the diagnosis and prognosis of renal neoplasms.

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.

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 angiotensin's 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.

Neoadjuvant chemotherapy modifies serum angiotensinase activities in women with breast cancer

PurposeThe aim of this study was to investigate the putative changes in serum angiotensinase activities (aminopeptidase N, APN; aminopeptidase B, APB; aminopeptidase A, APA; aspartyl aminopeptidase, ASAP) involved in the renin–angiotensin system (RAS) in women with breast cancer treated or not with a neoadjuvant therapy of paclitaxel and anthracycline and in healthy women volunteers. MethodsWe fluorometrically analysed serum APN, APB, APA and ASAP activities using their corresponding aminoacyl-β-naphthylamides as substrates in women with breast cancer treated with a neoadjuvant therapy of paclitaxel and anthracycline. ResultsWhen compared with healthy controls, women with breast cancer not treated with neoadjuvant chemotherapy, showed a decrease in angiotensinase activity, which support the putative increase of angiotensin II (Ang II) levels, indicating that the tumour process would favour the development of the disease. Also, an increase in APN and APB activities was observed, which support a role for angiotensin IV (Ang IV). In women treated with a neoadjuvant therapy, we described an increase in ASAP and APA activities, supporting the idea that this treatment increases Ang II catabolism. The resulting decrease in Ang II level could lead to an inhibition of the tumour growth. ConclusionPresent results show changes in serum angiotensinase activities in women with breast cancer and in women with breast cancer treated with a neoadjuvant therapy of paclitaxel and anthracycline. Therefore, considerable attention should be focused on the development of RAS blockade therapy as a new strategy for breast cancer treatment.

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.

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.

Blood pressure increased dramatically in hypertensive rats after left hemisphere lesions with 6-hydroxydopamine

Plasma angiotensinase activity, nitric oxide and systolic blood pressure (SBP) were differently affected after unilateral intrastriatal injection of 6-hydroxydopamine (6-OHDA), depending on the brain hemisphere injured. Moreover, normotensive and hypertensive rats responded differently suggesting an asymmetry in the organization of the autonomic nervous system of the vessels. The aim of this study was to investigate the evolution of SBP and heart rate (HR) over time after nigrostriatal lesions in normotensive and hypertensive rat strains. Unilateral depletions of brain dopamine were performed by injecting 6-OHDA into the left or right striatum of normotensive and hypertensive rats. Vehicle without 6-OHDA was unilaterally injected in control (sham) groups. SBP and heart rate (HR) were measured in un-anesthetised animals 10 and 3 days before administration of 6-OHDA or vehicle and 3 and 25 days after treatment. In normotensive rats, at the end of study, SBP increased significantly from pre-lesioned values in left-lesioned animals but no differences were observed in right-lesioned or sham groups. Before sacrifice, there was a significant reduction from pre-lesion values in HR. In hypertensive animals, there was a highly significant increase of SBP in left-lesioned and sham left rats and a slight increase in right-lesioned but no differences were observed in sham right group. No differences in HR were observed throughout the study in the groups studied. The present results represent direct experimental evidence of an asymmetrical cardiovascular response to unilateral brain lesions, suggesting that left injury may have a worst prognosis.

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.

Correlations between angiotensinase activity asymmetries in the brain and paw preference in rats

The function of angiotensin peptides is dependent upon the action of several aminopeptidases (APs) termed angiotensinases. Soluble (SOL) and membrane (MEM)-bound alanyl-AP (AlaAP) and cystinyl-AP (CysAP) are involved in the metabolism of angiotensins and related to the modulation of behavior and memory. To study the interactions between angiotensinase activity in the hippocampus and behavioral lateralization, Wistar rats were selected on the basis of their performance in the paw preference test (left-handed, ambidextrous and right-handed) and the activities of SOL-AlaAP/CysAP and MEM-AlaAP/CysAP were measured in the both hippocampuses. We observed that: (1) the left hippocampus had higher activities of SOL-AlaAP/CysAP and MEM-AlaAP/CysAP than the right hippocampus; (2) rats showed significant differences in the activities of SOL-AlaAP/CysAP and MEM-AlaAP/CysAP in the hippocampus depending on the behavioral lateralization detecting by paw preference; (3) in three groups of rats, hemispheric dominance - %R/T [%R/T=right hemisphere/(right hemisphere+left hemisphere)x100] activities of MEM-AlaAP, SOL-CysAP and MEM-CysAP was significantly different whereby %RT was lower in left-handed, higher in ambidextrous and intermediate in right-handed rats; (4) individual %R/T activities of SOL-CysAP and MEM-CysAP in the hippocampus were positively correlated with paw preference scores. Finally, we used the passive avoidance behavior test to demonstrate the differences of long-term memory among the three groups. These results suggested that the asymmetric activity of angiotensinase in the rat hippocampus may be associated with both the direction and the intensity of behavioral lateralization as expressed by paw preference.

Development of a homogeneous immunoassay for the detection of angiotensin I in plasma using AlphaLISA acceptor beads technology

Plasma renin activity (PRA) is a well-established biomarker for assessing the efficacy of various antihypertensive agents such as direct renin inhibitors, angiotensin receptor blockers, and angiotensin-converting enzyme inhibitors (ACEIs). PRA measurements are obtained through the detection and quantification of angiotensin I (Ang I) produced by the action of renin on its natural substrate angiotensinogen. The most accepted and reproducible method for PRA measurement uses an antibody capture Ang I methodology that employs specific antibodies that recognize and protect Ang I against angiotensinase activities contained in plasma. The amount of Ang I is then quantified by either radioimmunoassay (RIA) or enzyme immunoassay (EIA). In the current report, we describe the optimization of a novel homogeneous immunoassay based on the AlphaScreen technology for the detection and quantification of antibody-captured Ang I using AlphaLISA acceptor beads in buffer and in the plasma of various species (human, rat, and mouse). Ex vivo measurements of renin activity were performed using 10 μl or less of a reaction mixture, and concentrations as low as 1 nM Ang I were quantified. Titration curves obtained for the quantification of Ang I in buffer and plasma gave similar EC 50 values of 5.6 and 14.4 nM, respectively. Both matrices generated an equivalent dynamic range that varies from approximately 1 to 50 nM. Renin inhibitors have been successfully titrated and IC 50 values obtained correlated well with those obtained using EIA methodology ( r 2 = 0.80). This assay is sensitive, robust, fast, and less tedious than measurements performed using nonhomogeneous EIA. The AlphaLISA methodology is homogeneous, does not require wash steps prior to the addition of reagents, and does not generate radioactive waste.

Effect of Acetaldehyde Upon Cathepsin G and Chymase. NRAS Implications

Hypertension is commonly observed in alcoholics. Both the renin-angiotensin system (RAS) and the non-renin-angiotensin system (NRAS) have been implicated in the dynamics of blood pressure maintenance. In bilaterally nephrectomized rats, acetaldehyde has been reported to enhance the generation of the rate-limiting angiotensin I (ANG I) in the plasma, and in humans it inhibits the activity of several angiotensinases (A, B, and M) in the serum, thereby promoting a hypertensive set of reactions. We report here the results of a study on the effect of acetaldehyde upon cathepsin G and mast cell chymase. Acetaldehyde enhanced cathepsin G activity at all of the concentrations tested between 11.2 and 223.5 mM in a statistically significant manner. Since cathepsin G is one of several enzymes transforming ANG I into ANG II and is also capable of cleaving ANG II directly from angiotensinogen, we suggest that alcoholism, which will generate exogenous acetaldehyde from ingested alcohol, may be a contributory factor for an elevated cathepsin G activity and, consequently, hypertension via the NRAS. Chymase activity also is elevated in the presence of 440 mM acetaldehyde and diminished in the presence of 27 mM acetaldehyde. Since both enzymes also degrade ANG II, the degradative effects of each enzyme on ANG II may neutralize one another.

Atrial Angiotensinase Activity in Hypothyroid, Euthyroid, and Hyperthyroid Rats

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 microg/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.