Poster
# 76
Poster |
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6th Internet World Congress for Biomedical Sciences |
María Jesús García-López(1), María Jesús Ramírez-Expósito(2), José Manuel Martínez-Martos(3), María Dolores Mayas-Torres(4), Isabel Prieto-Gómez(5), Garbiñe Arechaga-Maza(6), Manuel Ramírez-Sánchez(7)
(1)(3)(4)(5)(6)(7)Unit of Physiology. University of Jaén - Jaén. Spain
(2)Unit of Physiology. University of Jaen - Jaén. Spain
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[Biochemistry] |
[Endocrinology] |
[Neuroscience] |
[Physiology] |
[Reproduction Sciences] |
Aminopeptidases are generally zinc-metalloenzymes that have been used in clinical chemistry as serum markers for several diseases, and which also play a physiological role in the regulation of circulating biologically active peptides. Hormonal changes in the organism may be reflected in aminopeptidase activity. However, although their hydrolytic action on peptides or artificial substrates has been extensevely studied, the actual physiological role of these enzymes and their own mechanism of regulation are not well known.
Previous studies have suggested an influence of gonadal steroids on serum AP activities (Gandarias et al., 1989; Martínez et al., 1997). We studied the possible influence of orchiectomy and testosterone on aminopeptidase A activity (Aspartyl aminopeptidase and glutamyl aminopeptidase).
In this work, forty male mice Balb-C mice were used (30.575 g body weight). The animal were randomly divided into 5 groups of 8 mice each one. All the animals had free access to fed and water and were housed at a constant temperature of 25 ºC with lights on from 7:00 am to 7:00 pm. Four groups of mice were orchiectomized and the fifth group was used as controls. Fifteen days after gonadectomy, three of this orchiectomized group were injected with a testosterone solution (2 mg/ml), in an increasig doses during ten days. The concentration administred to the groups were 1, 2 and 3 mg of testosterone respectively (E1, E2 and E3). The fourth group orchiectomized (ORX) and the control were only injected with 100 µl of sesame oil, used as vehicle. After that, all animals were sacrificed. The animals were anaesthetized by an intraperitoneal injection of equithesin. Blood samples were obtained from the left cardiac ventricle and hypophysis, hypothalamus, frontal cortex and adrenal glands were also obtained. Blood samples were measured the same day and the tissues were frozen to -80ºC until its measure.
Tissue samples were homogenized in 10 volumes of 10mM HCl-Tris buffer (pH 7.4) and ultracentrifuged at 100 000 g for30 min (4 ºC) to obtain the soluble rraction. The resulting supernatants were used to measure soluble enzymatic activity (Sol) and protein content, assayed in triplicate. To solubilize membrane proteins, the pellets were rehomogenized in HCl-Tris buffer (pH 7.4) plus 1% Triton X-100. After centrifugation (100 000g, 30 min, 4 ºC) the supernatants were used to measure membrane-bound activity (M-B) and proteins, also in triplicate. To ensure complete recorvery of activity, the detergent was removed from the medium by adding adsorbent polymeric Biobeads SM-2 (100 mg/ml) and shaking the samples for 2 h at 4 ºC.
GluAP activity was measured in a fluorimetric assay using Glutamyl- -naphthylamide (GluNNap), in accordance with the method of Tobe et al., (1980), with modifications. Asp AP activity was measured in a fluorimetric assay too, using Aspartyl- -naphthylamide following the method of Cheung and Cushman (1971). The amount of -naphthylamine released as a result of the enzimatic activity was measured fluorimetrically at an emission wavelength of 412 nm and an excitation wavelength of 345 nm. Proteins were quantified in triplicate by the method of Bradford (1976), using BSA as a standar. Specific Sol an M-B aminopeptidase activities were expressed as pmol of pGluNNap hydrolyzed per min per mg of protein. Fluorogenic assays were linear with respect to time of hydrolysis and protein content. We used one-way analysis of variance (ANOVA) to analyze differences between groups. Post-hoc comparisons were made using the paired Student´s t test; P values below 0.05 were considered significant.
Figure 1 shows Soluble and membrane bound AspAP activity. In serum, an increase of activity was observed after the injection of doses 1 (p<0.05; p<0.01) and 2 (p<0.01) of testosterone in relation with control group and ORX. Orchiectomy only reduce Sol AspAP activity in frontal cortex (p<0.01). Rest of tissues did not modified their activity. The levels of activity in frontal cortex after the injection of testosterone was maintained lower than in controls. The injection of dose 1 of testosterone increase significantly (p<0.01) the AspAP activity in hypophysis and hypothalamus. No changes were observed in membrane bound AspAP activity after orchiectomy.
Figure 2 shows soluble and membrane bound GluAP activity. In serum orchiectomy did not modified this activity, but the injection of doses 1 and 2 of testosterone increases significantly (p<0.01) GluAP activity. Soluble GluAP activity was decreased after orchiectomy in frontal cortex (p<0.05) and hypothalamus (p<0.05) and increased in hypophysis (p<0.01). The injection of testosterone reduce significantly (p<0.01) the levels of activity in hypothalamus. Membrane-bound GluAP activity was increased (p<0.01) after orchiectomy in Hypophysis but no changes were observed in rest of tissues.
We found significant differences in AP A activity in serum and different periferic tissues. Moreover, our results also demonstrate an influence of orchiectomy and testosterone on AP A activity. These enzymes responded in a different way in the analized situations. Testosterone increases AspAP and GluAP activities in serum, but in the other tissues the behaviour of these enzymes was quite different. Soluble AspAP only decreased in FC, the rest of tissues did not change with orchiectomy or testosterone. However, Sol GluAP activity suffered more modifications. These results may indicate that Asp- and GluAP are different enzymes although their action may be very simillar.
By other hand, AP activity plays a major role in the regulation of circulating biologically active peptides (Sanderik et al., 1988). Aminopeptidase A Appears to be a membrane-bound ectoenzime especially abundant in the kidney (Lodja and Gossrau, 1980) and is also found in the vascular circulation, where it appears to originate from the autolysis of membrane-bound AP A (Wilk and Healy, 1993). This is analogous to angiotensin-converting enzyme, which is originally membrane-bound but becomes soluble in plasma with autolysis (Williams, 1988). In any case, it is unlikely that the major mechanism of regulation of AP activity performs through active synthesis or release. Passive regulation of circulating and tissullar AP is plausible, depending on the biochemical enviroment, under specific cphysiological or pathological conditions. Our results are in accordance with that theory, because indicate the existence of an hormonal influence on AP activity. However, due to the role of AP A on the systemic and local renin-angiotensin systems, hormonal modifications during physiological and/or pathological conditions could modify blood pressure regulation through these systems.
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[Biochemistry] |
[Endocrinology] |
[Neuroscience] |
[Physiology] |
[Reproduction Sciences] |
