Poster
# 75
Poster |
|
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
|
 |
|
|
|
|
[Biochemistry] |
[Endocrinology] |
[Neuroscience] |
[Physiology] |
Aminopeptidases (AP) are generally zinc-metalloenzimes distributes in tissues and corporal fluids. These AP hydrolized the amino-terminal extreme of active peptides and polipeptides, so AP play a physiological role in the regulation of circullating biologically active peptides (Mc Donald and Barret, 1986; Sanderik et al., 1988). Therefore, hormonal changes in serum may be reflected in thes enzymatic activity. However, although their hydrolitic action on peptides or artificial substrates has been extensively studies. The actual physiological role of these enzymes and their own mechanism of regulation are not well known. Previous results have suggested an influence of gonadal steroids on serum AP activities (Gandarias et al., 1989;Martínez et al., 1997) raising the possibility that such substances help to create a biochemical enviroment that regulate, in part, the activity of these enzimes. One of these aminopeptidases is pyroglutamil aminopeptidase (pGluAP). pGluAP can be classified as an omega-peptidase which removes pyroglutamyl N-terminal residues from peptides, proteins and arylamidases derivaties in a highly selective manner (1,2). This enzime may be implicated in the regulation of several process in the CNS, and thyrotropin-releasing hormone (TRH) is considered as a possible endogenous substrate.
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.
pGluAP activity was measured in a fluorimetric assay using L-Pyroglutamyl- -naphthylamide (pGluNNap), in accordance with the method of Schwabe and McDonald (1977), 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 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 the mean±SEM of serum and soluble and membrane bound pGluAP activity in adrenal glands, hypophisis, frontal cortex and hypothalamus in all the experimental groups considered.
In serum orchiectomy caused a increase of activity (p<0.05). Testosterone in E1 and E2 did not change the activity, however, pGluAP activity in E3 group decreased significantly (p<0.01) in relation with orchiectomized mice.
In adrenal glands soluble pGluAP activity did not change with orchiectomy. This activity changed after the injection of dose 1. E1 group was significantly lower than control (p<0.05). No variations were observed between control groups and rest of all the groups in membrane bound pGluAP.
In hypophisis, soluble pGluAP activity increased significantly (p<0.05) after orchiectomy. This activity in E1 group was higher than in controls (p<0.01) and than in ORX (p<0.05). A decrease dose-dependent was observed in pGluAP activity after the injection of doses 2 and 3. membrane bound activity did not show differences after orchiectomy and after injection of testosterones.
In frontal cortex, orchiectomy did not induce differences in soluble pGluAP activity. The injection of testosterone neither produced changes in soluble pGluAP activity. In membrane bound pGluAP activity, no changes were observed after orchiectomy. E3 groups is the oly group where membrane bound pGluAP activity was modified. Lower levels of activity in relation with control (p<0.01) and ORX (p<0.05) was observed in E3 group.
In Hypothalamus, in E2 groups, soluble pGluAP activity was higer than in controls and in ORX group (p<0.05). No changes were observed betwee the other groups. Membrane bound pGluAP activity was similar in all the experimental groups. Orchiectomy and injection of testosterone did not induce differences between the groups.
We report here the influence of orchiectomy and testosterone on pyroglutamyl- -naphthylamide hydrolising activity levels in serum and different tissues in mice. pGluAP is an omega peptidase which removes pyroglutamyl N-terminal residues from peptides and arylamide derivatives. This enzime is widely distributed in fluids and tissues. To date, three distint types of this enzime have been described and called serum thyroliberinase , cytosolic pyroglutamil aminopeptidase type I and membrane bound pyroglutamyl aminopeptidase type II. The activity of all of them is thought to be implicated in the regulation, more or less restricted in their substrate specificity, of various susceptible endogenous substrates such as TRH, GnRH, neurotensin and bombesin (Bauer, 1994; Browne and O´Cuinn,1983; Cummins and O´Connor, 1996; Taylor and Dixon, 1978).
Our results suggest that orchiectomy and posterior injection of testosterone modified the pGluAP activity not only in serun but also in different periferic tissues. These results also suggest that the hormonal status in the animal may modificate the action of pGluAP, so it is possible that the metabolism of endogenous substrates as TRH may be affected by the hormonal microenviroment exists in the animals. However, the existence of changes in adrenal status affect TRH in parallel with corticopropin releasing hormone (Kakucska et al., 1995). Moreover, the potential pyroglutamyl aminopeptidase activity on N-terminal pGlu residues other than TRH should be taken into accounts as well.
These results may demostrate a direct or indirect relation between steroid hormones on pGluAP activity. and also suggested that the metabolism of some hormones such as TRH may be modified by the hormonal status.
|
|
|
|
|
|
|
|
[Biochemistry] |
[Endocrinology] |
[Neuroscience] |
[Physiology] |
