Correlational Study of Aminopeptidase Activities between Left or Right Frontal Cortex versus the Hypothalamus, Pituitary, Adrenal Axis of Spontaneously Hypertensive Rats Treated with Hypotensive or Hypertensive Agents.

It has been suggested that the neuro-visceral integration works asymmetrically and that this asymmetry is dynamic and modifiable by physio-pathological influences. Aminopeptidases of the renin-angiotensin system (angiotensinases) have been shown to be modifiable under such conditions. This article analyzes the interactions of these angiotensinases between the left or right frontal cortex (FC) and the same enzymes in the hypothalamus (HT), pituitary (PT), adrenal (AD) axis (HPA) in control spontaneously hypertensive rats (SHR), in SHR treated with a hypotensive agent in the form of captopril (an angiotensin-converting enzyme inhibitor), and in SHR treated with a hypertensive agent in the form of the L-Arginine hypertensive analogue L-NG-Nitroarginine Methyl Ester (L-NAME). In the control SHR, there were significant negative correlations between the right FC with HPA and positive correlations between the left FC and HPA. In the captopril group, the predominance of negative correlations between the right FC and HPA and positive correlations between the HPA and left FC was maintained. In the L-NAME group, a radical change in all types of interactions was observed; particularly, there was an inversion in the predominance of negative correlations between the HPA and left FC. These results indicated a better balance of neuro-visceral interactions after captopril treatment and an increase in these interactions in the hypertensive animals, especially in those treated with L-NAME.

Hypothalamic Renin-Angiotensin System and Lipid Metabolism: Effects of Virgin Olive Oil versus Butter in the Diet.

The brain renin-angiotensin system (RAS) has been recently involved in the homeostatic regulation of energy. Our goal was to analyse the influence of a diet rich in saturated fatty acids (butter) against one enriched in monounsaturated fatty acids (olive oil) on hypothalamic RAS, and their relationship with the metabolism of fatty acids. Increases in body weight and visceral fat, together with an increase in aminopeptidase A expression and reductions in AngII and AngIV were observed in the hypothalamus of animals fed with the butter diet. In this group, a marked reduction in the expression of genes related to lipid metabolism (LPL, CD36, and CPT-1) was observed in liver and muscle. No changes were found in terms of body weight, total visceral fat and the expression of hepatic genes related to fatty acid metabolism in the olive oil diet. The expressions of LPL and CD36 were reduced in the muscles, although the decrease was lower than in the butter diet. At the same time, the fasting levels of leptin were reduced, no changes were observed in the hypothalamic expression of aminopeptidase A and decreases were noted in the levels of AngII, AngIV and AngIII. These results support that the type of dietary fat is able to modify the hypothalamic profile of RAS and the body energy balance, related to changes in lipid metabolism.

Functional and neurometabolic asymmetry in SHR and WKY rats following vasoactive treatments.

A lateralized distribution of neuropeptidase activities in the frontal cortex of normotensive and hypertensive rats has been described depending on the use of some vasoactive drugs and linked to certain mood disorders. Asymmetrical neuroperipheral connections involving neuropeptidases from the left or right hemisphere and aminopeptidases from the heart or plasma have been suggested to play a role in this asymmetry. We hypothesize that such asymmetries could be extended to the connection between the brain and physiologic parameters and metabolic factors from plasma and urine. To assess this hypothesis, we analyzed the possible correlation between neuropeptidases from the left and right frontal cortex with peripheral parameters in normotensive (Wistar Kyoto [WKY]) rats and hypertensive rats (spontaneously hypertensive rats [SHR]) untreated or treated with vasoactive drugs such as captopril, propranolol and L-nitro-arginine methyl ester. Neuropeptidase activities from the frontal cortex were analyzed fluorometrically using arylamide derivatives as substrates. Physiological parameters and metabolic factors from plasma and urine were determined using routine laboratory techniques. Vasoactive drug treatments differentially modified the asymmetrical neuroperipheral pattern by changing the predominance of the correlations between peripheral parameters and central neuropeptidase activities of the left and right frontal cortex. The response pattern also differed between SHR and WKY rats. These results support an asymmetric integrative function of the organism and suggest the possibility of a different neurometabolic response coupled to particular mood disorders, depending on the selected vasoactive drug.

Enkephalinase activity is modified and correlates with fatty acids in frontal cortex depending on fish, olive or coconut oil used in the diet.

OBJECTIVE: Enkephalins are neuropeptides involved in functions such as pain modulation and/ or cognitive processes. It has been reported that dietary fat modifies enkephalins in the brain. Since enkephalins are hydrolyzed by enkephalinases, the study of the influence of dietary fats, differing in their degree of saturation, on brain fatty acids content and enkephalinase activity is important to understand its regulatory role on neuropeptides under different type of diets. METHODS: We analyzed enkephalinase activity, assayed with alanine-ß-naphthylamide as sub-strate, in frontal cortex of adult male rats fed diets supplemented with fish oil, olive oil or coconut oil, which markedly differed in the saturation of their fatty acids. RESULTS: Rats fed a diet enriched with coconut oil had lower soluble enkephalinase activity than the group fed olive oil (p<0.01) and fish oil (p<0.05) whereas rats fed a diet enriched with fish oil had lower membrane-bound enkephalinase activity than the group fed with olive (p<0.001) or coconut oil (p<0.05). Significant negative correlations were observed between certain fatty acids and enkephalinase activities in the groups fed with olive and coconut oils. No correlations were observed in the group fed with fish oil. CONCLUSIONS: Dietary fat modifies enkephalinase activity in the frontal cortex depending on the degree of saturation of the used oil. It is postulated that the functions, in which enkephalins are involved, such as pain modulation or cognitive functions, may also be affected according to the type of oil used in the diet.

Divergent profile between hypothalamic and plasmatic aminopeptidase activities in WKY and SHR. Influence of beta-adrenergic blockade.

AIMS: Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) differ in their renin-angiotensin system function and sympathetic tone. The metabolism of angiotensins and vasopressin depends on the action of certain aminopeptidases whose activity may be influenced by the autonomic nervous system. Their regulation may differ between WKY and SHR in hypothalamus and plasma according to the sympathetic tone. We analyzed aminopeptidases responsible for the hydrolysis of certain angiotensins, vasopressin, cholecystokinin or enkephalins in hypothalamus and plasma of WKY and SHR in untreated controls rats and under beta-adrenoceptor blockade. Systolic blood pressure, food intake, water intake and diuresis were measured as parameters modulated by the autonomic nervous system and the above mentioned peptides. MAIN METHODS: Glutamyl-, aspartyl-, cystinyl- and alanyl-aminopeptidase activities were analyzed fluorimetrically in plasma and hypothalamus of control and propranolol-treated (100mg/kg/day administered in drinking water for 1month) WKY and SHR, using arylamide derivatives as substrates. KEY FINDINGS: An opposite response of aminopeptidases to propranolol treatment between plasma and hypothalamus was observed in either WKY and SHR. Furthermore, the behavior of aminopeptidases was inversed between WKY and SHR either in hypothalamus and plasma: while the activity increased in hypothalamus and decreased in plasma of WKY, it decreased in hypothalamus and increased in plasma of SHR. SIGNIFICANCE: These results revealed an inverse response of aminopeptidases between hypothalamus and plasma and also an opposite behavior of these enzymes between WKY and SHR in hypothalamus and plasma. These observations support the involvement of the sympathetic system in the modulation of aminopeptidase activities.

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.

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.

Lipid oxidation inhibitory effects and phenolic composition of aqueous extracts from medicinal plants of Colombian Amazonia.

Diverse plants of ethnobotanic interest in Amazonia are commonly used in traditional medicine. We determined the antioxidant potential against lipid peroxidation, the antimicrobial activity, and the polyphenol composition of several Amazonian plants (Brownea rosademonte, Piper glandulosissimum, Piper krukoffii, Piper putumayoense, Solanum grandiflorum, and Vismia baccifera). Extracts from the plant leaf, bark, and stem were prepared as aqueous infusions, as used in folk medicine, and added to rat liver microsomes exposed to iron. The polyphenolic composition was detected by reverse-phase HPLC coupled to diode-array detector and MS/MS analysis. The antimicrobial activity was tested by the spot-on-a-lawn method against several indicator microorganisms. All the extracts inhibited lipid oxidation, except the P. glandulosissimum stem. The plant extracts exhibiting high antioxidant potential (V. baccifera and B. rosademonte) contained high levels of flavanols (particularly, catechin and epicatechin). By contrast, S. grandiflorum leaf, which exhibited very low antioxidant activity, was rich in hydroxycinnamic acids. None of the extracts showed antimicrobial activity. This study demonstrates for the first time the presence of bioactive polyphenolic compounds in several Amazonian plants, and highlights the importance of flavanols as major phenolic contributors to antioxidant activity.

Characterization of Enterococcus faecalis and Enterococcus faecium from wild flowers.

Wild flowers in the South of Spain were screened for Enterococcus faecalis and Enterococcus faecium. Enterococci were frequently associated with prickypear and fieldpoppy flowers. Forty-six isolates, from 8 different flower species, were identified as E. faecalis (28 isolates) or E. faecium (18 isolates) and clustered in well-defined groups by ERIC-PCR fingerprinting. A high incidence of antibiotic resistance was detected among the E. faecalis isolates, especially to quinupristin/dalfopristin (75%), rifampicin (68%) and ciprofloxacin (57%), and to a lesser extent to levofloxacin (35.7%), erythromycin (28.5%), tetracycline (3.5%), chloramphenicol (3.5%) and streptomycin (3.5%). Similar results were observed for E. faecium isolates, except for a higher incidence of resistance to tetracycline (17%) and lower to erythromycin (11%) or quinupristin/dalfopristin (22%). Vancomycin or teicoplanin resistances were not detected. Most isolates (especially E. faecalis) were proteolytic and carried the gelatinase gene gelE. Genes encoding other potential virulence factors (ace, efaA (fs), ccf and cpd) were frequently detected. Cytolysin genes were mainly detected in a few haemolytic E. faecium isolates, three of which also carried the collagen adhesin acm gene. Hyaluronidase gene (hyl ( Efm )) was detected in two isolates. Many isolates produced bacteriocins and carried genes for enterocins A, B, and L50 mainly. The similarities found between enterococci from wild flowers and those from animal and food sources raise new questions about the puzzling lifestyle of these commensals and opportunistic pathogens.

Semi-preparative scale purification of enterococcal bacteriocin enterocin EJ97, and evaluation of substrates for its production.

The influence of substrate composition on the production of enterocin EJ97 and the conditions for semi-preparative bacteriocin recovery have been studied. Final bacteriocin concentrations of 12.5 or 15.6 mg/l were obtained in the commercial media brain heart infusion broth (BHI) and tryptic soya broth, respectively. The bacteriocin was also produced in the complex medium CM (8.75 mg/l), in which the vitamin supplement was essential for production. Some combinations of meat peptone and yeast extract plus either soy peptone or BHI also supported bacteriocin production, at concentrations of 6.25-7.5 mg/l. In cow milk (whole, half-skimmed, and skimmed), the final bacteriocin concentrations obtained ranged from 7.5 to 11.25 mg/l. Highest bacteriocin activity was obtained by using pasteurised milk whey as growth substrate (up to 25 mg/l), suggesting that this bacteriocin can be obtained on a large scale by using this cheap food-grade industrial by-product. Highest bacteriocin titres were always obtained after 8 h of incubation at 37 degrees C. Semi-preparative concentration and purification of enterocin EJ97 produced in a complex medium was achieved by bulk cation exchange chromatography without previous cell separation, followed by reversed-phase chromatography. This two-step procedure allowed preparation of milligram quantities of purified bacteriocin, which is an improvement compared to purification procedures established for most other bacteriocins (35). The availability of purified enterocin EJ97 will facilitate other studies such as the elucidation of its molecular structure and its interaction with target bacteria.