Poster
# 77
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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[Reproduction Sciences] |
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[Biochemistry] |
[Endocrinology] |
[Neuroscience] |
[Physiology] |
Aminopeptidases (AP) are generally zinc-metalloenzymes which are thought to be involved in the metabolism of proteins an in the regulation of various peptide hormones (Sanderik et al., 1988). Previous results have suggested an influence of gonadal steroid on aminopeptidase activities (Gandarias et al., 1989; Martínez et al., 1997). In present the present work we analyzed the effects of orchiectomy and injection of different doses of testosterone on different AP activities. We measured alnyl aminopeptidase (E.C. 3.4.11.14) (AlaAP), arginyl aminopeptidase (EC 3.4.11.6) (ArgAP) and cystinyl aminopeptidase (EC 3.4.11.3) (CysAP) which have been reported to be active on several circulating and tissue peptides (McDonald and Barret, 1986).
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.
AlaAP, ArgAP and CysAP activities were measured in a fluorimetric assay using Ala- -naphthylamide (AlaNNap), Arg- -naphthylamide (ArgNNap) and Cys- -naphthylamide as substrates, in accordance with the method of Greeberg (1962), with modifications. 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 AlaNNap, ArgNNap and CysNNap 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.
The influence of orchiectomy and testosterone on Ala-, Arg-, and CysAP activities shows the following results:
In serum, AlaAP and CysAP did not change, but ArgAP activity decreased after orchiectomy. The injection of testosterone induced an increase in ArgAP activity. In frontal cortex, orchiectomy produced a significant decrease in soluble CysAP only. Testosterone induced a significant increase in soluble AlaAP. Higher soluble ArgAP and CysAP activities were observed after injection od doses E2 and E3. In hypothalamus, the soluble aminopeptidases analyzed, did not change after orchiectomy, but the injection of dode E2 of testosterone produced significant increases in soluble AlaAP, ArgAP and CysAP. However, membrane bound AlaAP, ArgAP and CysAP activities increase in orchiectomized group. Significant changes were observed in these AP activities after injection of testosterone. Soluble Ala, Arg and CysAP were increased in hypophysis in all the group analized. In adrenal glands, no changes were observed in soluble and membrane bound Ala, Arg and Cys AP activities after orchiectomy, but differences were observed between E1, E2 and E3 groups and control animals.
We found significant changes of AP activities in specific tissues, although in serum no changes were described. Previous preliminary in vitro studies using water-soluble complexes with cyclodextrines as carried showed that steroids hormones influence AP activities (Gandarias et al., 1989; Ramírez et al., 1993).
In addition, aminopeptidase B like activity (ArgAP) has been isolated from rat testes and has been implicated in several steps of spermatogenesis (Cadel et al., 1995). Therefore, the present results represent additional suport of a steroid influence on AP activities. We observed changes in AlaAP and ArgAP activities. Enkephalins, which play a neuromodulatory role in brain, have been described as substrates for both activities (Johnson and Hersh, 1990). Futhermore, AP activity plays a major role in the regulation of several biologically active peptides in circulation and tissues. AlaAP may hydrolise bradykinins (Sanderik et al., 1988) and enkephalins (Hersh, 1985) and may also act as an angiotensinase (Ahmad and Ward;1990). ArgAP activity specifically hydrolises basic N-terminal residues from peptides and arylamide derivaties (McDonald and Rarret, 1986). Because of its exopeptidase activity, it has been implicated in the metabolism of Met-enkephalin ((Johnson and Hersh, 1990) and angiotensin III (Ahmad and Ward, 1990); its endopeptidase activity is also thought to be involved inneurotensin metabolism ((McDermott et al., 1988). CysAP activity has been reeported to hydrolise oxytocin and vasopressin (Itoh and Nagamutsu, 1995), Therefore, the present results demonstrate that orchiectomy and testosterone influence aminopeptidase activities in specific locations in mice. These effects may induce functional modifications in the action of aminopeptidases on susceptible substrates; altermatively, they may reflect concomitant modifications in susceptible endogenous substrates.
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[Reproduction Sciences] |
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[Biochemistry] |
[Endocrinology] |
[Neuroscience] |
[Physiology] |
