CHANGES IN MEMBRANE-BOUND LEUCINE AMINOPEPTIDASE ACTIVITY DURING MATURATION AND AGEING OF BRAIN

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1 pages CHANGES IN MEMBRANE-BOUND LEUCINE AMINOPEPTIDASE ACTIVITY DURING MATURATION AND AGEING OF BRAIN Garbifie Arechaga 1, Jos~ M. Martinez 2, Isabel Prieto 2, Maria J. Ramlrez 2, Fraucisco Mba 3 and Manuel Ramirez 2. 1Department if Physiology, Medical School University of the Basque Country, Leioa, Vizcaya, Spain 2Unit qf Phfsiology, Faculty qf ExperimentaI Sciences, Universi(T qf ]ac;n, E ]adn, Spain ~Department qf Biochemistrp and Molecular Biology, Medical School, University of Granada, Granada, Spain "To whom correspondence should be addressed Summary. Aminopeptidases are believed to be enzymes that regulate the activity of various neuropeptides. However, their physiological role, as well as their mechanisms of regulation, are not well understood. To analyze a part of the regulatory mechanisms that control the activity of these enzymes, the subcellular distribution of membrane-bound leucyl aminopeptidase activity was studied in rat brain during development and ageing. Except in fetuses, the enzymic activity was greatest in the microsomal fraction in all ages tested. Except in microsomal and myelin fractions, compared with fetuses, leucyl aminopeptidase activity showed a decrease in l-weekold rats and a subsequent increase to adult levels in 1-month-old rats. This profile differed in the microsomal fraction, where the activity increased steadily up to 1-month-old rats. After this age, the activity decreased progressively in 5-month and 24-month-old rats. These results may reflect changes in the functional status of the susceptible substrates during development and ageing. Key words: development, ageing, subcellular fractionation, leucine aminopeptidase, rat brain INTRODUCTION It is currently believed that neuropeptide function is essentially regulated by the action of proteolytic enzymes. Aminopeptidases play a major role in this regulation and have been involved in the metabolism of several neuropeptides (1). These enzymes do not simply inactivate a peptide but may modify its biological activity (2). The arylamidases represent a subclass of aminopeptidases that hydrolyse amino acid 13-naphthylamides in addition to peptide substrates. These enzymes have also been implicated in the metabolism of a variety of physiologically active peptides, and their study promises to shed light on neuropeptide function (3). Leucyl aminopeptidase activity (LeuAP) (E.C ) has been demonstrated to be active on enkephalins (3), dynorphins (4) and Substance P (5), and also hydrolyses arylamide derivatives (1). However, the identification of a neuropeptide degrading activity itself is not a Copyright IUBMB /99 $

2 sufficient criterion for deducing that these enzymes have a biological function and their role is not well defined. To better understand the mechanisms which regulate the intracellular and extracellular concentrations of those peptides, it is important to determine the subcellular localization of the neuropeptide degrading enzymes (6). In addition, determination of any changes that occur during development and ageing, could contribute to clarifying the regulatory mechanism that controls the function of the susceptible substrates of these enzymes. To study the functional role of LeuAP activity, we analysed the subcellular distribution of membranebound LeuAP activity (M-B LeuAP) in rat brain during development and ageing, using Leu-[3- naphthylamide as substrate. MATERIALS AND METHODS Fetuses (19-20 d); 1-week-old; and 1-, 5- and 24-month-old male Sprague-Dawley rats were used in this study. The rats were housed at a constant temperature of 25 ~ with light on from 7:00 to 19:00 and free access to water and food. To avoid diurnal variations (7), all the experiments were started at the same time of the day (9:00). The experimental procedures for animal use and care were in accordance with the ethical principles and guidelines for scientific experiments on animals of the Swiss Academy of Medical Sciences. To determine fetal age, one male and six female adult rats were kept in a cage overnight and then the male was removed and gestation determined by the presence of sperm in vaginal smears. The fetuses were removed days later. Brains were perfused with saline through the left ventriculum under equithensin anaesthesia (2 ml/kg body wt). To obtain enough tissue for each experiment (2.5-3 g), the brains of 8- fetuses, five animals of 1 week, or three animals of 1, 5 and 24 months of age were pooled and used as starting material. After perfusion, the brains were quickly removed and homogenized in volumes of 0.32 M sucrose (homogenate). Subcellular fractions were obtained according to the method of Gray and Whittaker (8) modified by Krueger et al. (9). Briefly, from the crude mitochondrial pellet (P2) (12500 g), we obtained fractions A (myelin), B (synaptosomal) and C (mitochondrial). Fractions B, C and P3 (microsomal) (0000 g) and samples from the homogenate and crude nuclear pellet (P1) (00 g) were homogenized separately in Tris-HC1 mm (ph 7.4) and centrifuged (0000 g, 30 min, 4 ~ The resultant pellets and fraction A (myelin) were homogenized in Tris-HC1 mm (ph 7.4) plus 1% Triton X-0 to obtain, after centrifugation (0000 g, 30 min, 4 ~ supernatants that were used to test membrane-bound activity and proteins in triplicate. LeuAP was determined fluorometrically with Leu-fl-naphthylamide (LeuNNap) as substrate (). Proteins were measured in triplicate by the method of Bradford (11). Specific membrane-bound activities were expressed as nmol LeuNNap hydrolysed per min per mg protein. For comparison, we used one-way analysis of variance. If significant, the means were compared using the Newman-Keuls method. Values of p<0.05 were considered significant. RESULTS The levels of membrane-bound LeuAP specific activity, at the selected ages, in the different subcellular fractions of the rat brain, are shown in Fig

3 Homogenate 40 30[ Nuclear 2030 f ~ 20 0 I i J I r 0 F 1W 1M 5M 2Y I ~ I~N 1M 5~1 2,~M Microsomal " Myelin ~. 3o 301 "O 0 ~ I~N 1M 5Lt 2'~M 0 1~/ 1M 5M 24M E (- Synaptosomal Mitochondrial 3O 2O 20 0 i F i lw I i i 1M 5M 2Y F t i lw 1M 5M i 2Y 0 Fig. 1: Membrane-bound Leu-13-naphthylamide-hydrolyzing activity in homogenate, nuclear, microsomal, myelin, synaptosomal, and mitochondrial fractions of brain tissue from fetuses (F), 1-week-old (1W), 1-month-old (1M), 5-month-old (5M) and 24-month-old (24M) rats. Specific activity (Mean is expressed as nmol of Leu-13-naphthylamide hydrolyzed per min per mg protein (n=6-15); *P2 fraction in fetuses. The subcellular distribution of M-B LeuAP showed significant differences between fractions in all ages tested. In fetuses, the activity exhibited the lowest levels in the microsomal fraction (p<0.001). In contrast, 1-week, 1-month, 5-month and 24-month-old rats showed the highest levels in the microsomal fraction (p<0.001), whereas no differences were observed between the other fractions. Significant age-related changes were observed in all the fractions investigated. Compared with fetuses, a significant decrease was demonstrated in 1-week-old rats in homogenate (p<0.001), nuclear (p<0.05), synaptosomal (p<0.001) and mitochondrial (p<0.001) fractions, whereas no significant changes were observed in microsomal and myelin fractions. Except in 853

4 myelin, the fractions demonstrated a significant increase in 1-month-old rats compared with 1- week levels (p<0.01). Subsequently, a progressive decrease was observed. In 5-month-old rats, the activity exhibited a significant decrease in homogenate (p<0.001), microsomal (p<0.05), synaptosomal (p<0.001) and mitochondrial (p<0.05) fractions. Finally, compared with 5-month old rats, the 24-month-old ones displayed a significant reduction in homogenate (p<0.05), myelin (p<0.01) and mitochondrial (p<0.05) fractions. DISCUSSION Aminopeptidases play a role in processing and inactivation of neuropeptides and much work is devoted to clarifying their functions. Studies on development and localization are a common approach and these enzymes have exhibited age-related changes in central and peripheral tissues (12-14). Turner et al. (15) suggest that more than the specificity of peptidases for neuropeptides, their functional role depends on their subcellular localization and regional distribution. In this sense, soluble and membrane-bound Leu-aminopeptidase activities differ markedly in their regional distribution within the brain and their levels in cortex are inversely related to levels in subcortical brain areas. The soluble form of the enzyme activity is found throughout the brain, with only small differences among the various regions examined. However, membrane-bound LeuAP activity is distributed in a highly heterogeneous fashion. Most neuropeptides are concentrated in only a few regions of the brain. Thus, it appears unlikely that the role of soluble LeuAP is primarily to inactivate neuropeptides. It is more likely that membrane-bound LeuAP activity forms part of the mechanism of neuropeptide degradation (16). Therefore, in the present work, we studied the subcellular distribution of membrane-bound Leu-13-naphthylamidehydrolysing activity in rat brain during development and ageing. The results demonstrated that except in fetuses, the highest levels of M-B LeuAP activity appeared at microsomal level in the different age groups tested, which may be strongly related with the loci of synthesis and biotransformation of precursor peptides or the axoplasmic transport of proteins. The general increase between one week and one month of age and the decrease at older ages appear to parallel axonal and dendritic development during the first week post partum and dendritic and neuronal loss in aged animals (17). These changes are compatible with a role in synaptic events, in the control of activity of susceptible peptides. Moreover, the differences between ages suggest a significant change in the functional role of the susbstrates. In this connection, a significant decrease in the content of enkephalins was found in the basal ganglia of 854

5 22-month-old rats compared with 3-month-old animals (18). This observation is compatible with the decrease in activity in our aged animals. In this sense, the reduction of specific activity in adult and aged animals in all the fractions tested, is probably not due to differences in protein content since the most striking developmental changes in protein content take place before 30 days of age, and no significant differences are found at older ages (13,19). On the other hand, the microsomal fraction differed markedly from the other fractions: microsomal activity steadily increased from fetal to 1-month-old rats, whereas in the rest the activity decreased in 1-week-old rats and subsequently increased in 1-month-old animals. This suggests that, at the microsomal level, M-B LeuAP activity represents a major role in the biotransfbrmation of proteins and peptides during the end of the fetal period and the first stages of postnatal life. In addition, we have previously reported an influence of cholesterol on aminopeptidase activity (20); therefore, the significant decrease in 1-week-old rats observed in the present work could be related to cholesterol and phospholipid ontogeny in which it is clearly established that the content of these substances increases during the first stages of development to contribute to the building of cell membranes (21). At least three membrane-bound aminopeptidases have been characterized in the rat brain. Although aminopeptidase M I hydrolyses only the 13-naphthylamides of Arg and Lys, aminopeptidase M II and M exhibit a broad specificity with respect to amino acid 13- naphthylamides, including L-Leu-13-naphthylamide (22,23). Because aminopeptidase M activity comprises approximately 5 % of the total membrane aminopeptidase activity (22) we feel that the bulk of membrane-bound aminopeptidase activity, using L-Leu-13-naphthylamide as substrate, corresponds to aminopeptidase M II characterized by Hersh (23). Therefore, in view of the putative role of membrane-bound Leu-l~-naphthylamide-hydrolysing activity in enkephalindegrading aminopeptidase activity (23), it is of particular interest to note the age-related changes in the different fractions studied, which could reflect a functional modification in their action on susceptible substrates. In addition, according to Marks et al, (13), variation in the level of this enzyme with age may suggest differences in substrate specificity, based on modifications in its susceptible peptide substrates. REFERENCES 1. McDonald J. K. and Barret A. J. (1986) Leucyl aminopeptidase. In: Mammalian proteases: a glossary and bibliography. Vol 2, Academic Press, London, pp Lynch D. R. and Snyder S. H. (1986) Annu. Rev. Biochem. 55, Johnson G. D. and Hersh L. B. (1990) Arch. Bioehem. Biophys. 276,

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