Oxytocinase in the female rat hypothalamus: a novel mechanism controlling oxytocin neurones during lactation.

In addition to its peripheral actions, oxytocin released within the brain is important for birth and essential for milk ejection. The oxytocinase enzyme (placental leucine aminopeptidase; P-LAP) is expressed both peripherally and centrally. P-LAP controls oxytocin degradation in the uterus, placenta and plasma during pregnancy, although its role in the hypothalamus is unclear. We investigated P-LAP expression and activity in the hypothalamus in virgin, pregnant and lactating rats, as well as its role in vivo during the milk-ejection reflex. P-LAP mRNA and protein were expressed in magnocellular neurones of the supraoptic (SON) and paraventricular (PVN) nuclei. Oxytocin neurones co-expressed P-LAP without strong subcellular co-localisation of oxytocin and P-LAP, indicating that they are packaged in separate vesicles. Examination of the intracellular distribution of oxytocin and P-LAP showed a redistribution of P-LAP to within 1 µm of the plasma membrane in the somata of oxytocin neurones during lactation. Both P-LAP mRNA expression and hypothalamic leucyl/cystinyl aminopeptidase activity in the soluble fraction were higher during lactation than in late pregnant or virgin states. Inhibition of central enzyme activity by i.c.v. injection of amastatin in anaesthetised suckling mothers increased the frequency of reflex milk ejections. Because hypothalamic P-LAP expression and activity increase in lactation, and the prevention of its action mimics central oxytocin administration, we conclude that P-LAP regulates auto-excitatory oxytocin actions during the suckling-induced milk-ejection reflex.

Effects of dehydration on renal aminopeptidase activities in adult male and female rats.

Aminopeptidases (APs) are important regulators of peptides directly involved in water homeostasis such as angiotensins (Ang) and vasopressin (AVP). Sex differences in water balance and differences in the effects of gonadal steroids on osmotic stimulation of vasopressin secretion have been reported. Since sex steroids may be involved, the gonadotropin response to osmotic stimuli may be different between males and females. The purpose of this study was to determine the behavior of angiotensinases, vasopressin-degrading activity and gonadotropin-releasing hormone (GnRH)-degrading activity in the cortex and medulla of the kidney of dehydrated male and female rats. In the renal cortex, our results demonstrated an increase in Ang III-degrading activity in dehydrated males but not in females. This response may lead to an increased formation of Ang IV. This occurs with an increase in AspAP activity (which metabolizes Ang I to des-Asp(1)-Ang I), with no changes in Ang II-degrading activity and also with increased levels of AVP-degrading activity in dehydrated animals. These results may suggest an increased cortical blood flow due to enhanced formation of Ang IV together with reduced availability of the vasoconstrictor agents Ang II and AVP in the renal cortex of dehydrated males. The results obtained in the renal medulla suggest the inhibition of the metabolism of Ang I to des-Asp(1)-Ang I, together with a reduced metabolism of Ang II and AVP in dehydrated males but not in females. These results suggest a prolonged action of Ang II and AVP, which could stimulate sodium and water reabsorption in the medulla of dehydrated males. Changes in APs after dehydration occur preferentially in males, which may explain in part the reported sex differences in water homeostasis. The present results suggest a physiologically relevant role for AP activities in water homeostasis.

Effects of dietary supplementation with fish oil, lard, or coconut oil on oxytocinase activity in the testis of mice.

Oxytocin (OT), locally synthesized in the testis, is involved in androgen biosynthesis. The use of polyunsaturated fatty acids (e.g., fish oil) in the diet may improve the fertilizing ability in mammals. Cystinyl aminopeptidase (oxytocinase) activity plays a major role regulating the functional status of OT. Sex steroids and the type of the fatty acid used in the diet modify aminopeptidase activities in serum. In the present study, the authors compared the effect of a fish oil supplemented diet with two other diets supplemented with saturated oils (lard and coconut) on oxytocinase activity in the testis of mice. The enzymatic activity was determined fluorometrically using cystinyl-beta-naphthylamide as substrate. The results demonstrated higher levels of oxytocinase activity in mice fed the diet supplemented with fish oil than in those that were fed diets containing lard or coconut oils. The testicular functions in which OT is involved may be attenuated by the use of fish oil in the diet.

Environmental light-darkness conditions induce changes in brain and peripheral pyroglutamyl-peptidase I activity.

To evaluate the influence of light and darkness on brain pyroglutamyl-peptidase I (pGluPI) activity, four experimental groups of rats were compared at the same time-point (10.00 h). Two groups were designed with a standard 12-12 h light-dark cycle: In group A, the lights were on from 7.00 h to 19.00 h, and the experiment was done under light conditions; in group B, the lights were on from 19.00 h to 7.00 h, and the experiment was done under darkness conditions. Two additional groups were designed with nonstandard light-dark conditions: In group C, the animals were subjected to constant light, and the experiment was done under light conditions. In group D, animals were subjected to constant darkness, and the experiment was done under darkness conditions. Light (vs darkness) and standard (vs nonstandard) conditions produced significant changes on pGluPI activity in specific structures; the data suggested that endogenous substrates of pGluPI such as thyrotropin-releasing hormone and gonadotropin-releasing hormone, might be modified in parallel. There was left predominance in the retina under light conditions on a standard schedule (group A). The regional pattern of distribution of activity was similar in groups on a standard schedule (A vs B) and in groups tested under constant light-dark conditions (C vs D). However, this pattern differed between groups subjected to standard vs constant light-dark conditions (A and B vs C and D). These results support an influence of environmental light and darkness on pGluPI activity, which may reflect concomitant changes in its susceptible substrates and consequently in their functions.

Aminopeptidase activities after water deprivation in male and female rats.

Aminopeptidases (APs) play a major role in the metabolism of circulating and local peptides, such as angiotensins and vasopressin, substances involved in the control of blood pressure and water balance. In the present work, we studied the influence of dehydration on angiotensinases and vasopressin-degrading activity. Since sex differences may exist in the regulation of water balance by angiotensin II and differential sexual steroid modulation of vasopressin secretion, in response to osmotic stimulation have been reported, gonadotropin releasing hormone (GnRH)-degrading activity was also analysed in serum, neurohypophysis and adrenal glands of male and female rats. Our results did not suggest sex differences in the response to changes in osmolality. GnRH-degrading activity decreased in serum of dehydrated males and females, which suggests a longer action of the peptide under these conditions. In neurohypophysis, there was an increase in the activity of aminopeptidase A (APA), the enzyme responsible for the metabolism of angiotensin II to angiotensin III. This occurs with a decrease in alanyl aminopeptidase activity, which would lead to a prolonged action of angiotensin III by reduction of its metabolism. In adrenals of dehydrated animals, the results would imply a high degree of metabolism of angiotensin III and vasopressin.

Efecto del hipotiroidismo e hipertiroidismo sobre la actividad aminopeptidasa en plasma de ratas / Aminopeptidase activity and thyroid function in rats

Las alteraciones en la función del tiroides originan importantes cambios en la respuesta cardiovascular, en los que están implicados modificaciones en el sistema renina-angiotensina circulante (SRA) y otros péptidos vasoactivos. Las actividades aminopeptidasas (AP), a través del control de la hormona liberadora de la tirotropina (TRH), el SRA y otros péptidos vasoactivos como la vasopresina desempeñan un importante papel en el control de la función del tiroides y de la presión arterial. Con el fin de evaluar el papel de distintas aminopeptidasas plasmáticas en la función tiroidea, determinamos las actividades alanina (AlaAP), cistina (CysAP), piroglutamato (pGluAP), glutamato (GluAP) y aspartato (AspAP) aminopeptidasa, utilizando derivados de la naftilamida como sustratos en animales eu hipo e hipertiroideos. Los resultados demuestran que el hipertiroidismo disminuye significativamente las actividades pGluAP y CysAP, mientras que aumenta la actividad AlaAP.Sin embargo, no se observaron diferencias para las actividades AspAP y GluAP. El hipotirodismo incrementó significativamente los valores de AlaAP, no observándose diferencias en el resto de las actividades. Los presentes resultados apuntan a un papel preponderante de la actividad AlaAP (AP M) en lugar de la GluAP (AP A) en la regulación del SRA circulante, en modelos animales de hiper e hipotiroidismo (AU)

Serum aminopeptidase A activity of mice is related to dietary fat saturation.

A high intake of monounsaturated fat has been proposed to be a dietary factor that can decrease the incidence of cardiovascular disease and hypertension. In addition, increasing dietary fat saturation has been shown to increase plasma total cholesterol and elevate systolic and diastolic blood pressures. We demonstrated previously that cholesterol selectively increases in vitro aminopeptidase A activity, which is related to angiotensin metabolism. In this study, we investigated the effect of different degrees of dietary fatty acid saturation on serum aminopeptidase activities in vivo. Serum total cholesterol concentrations were also measured. Five groups of male Balb/C mice were fed for 10 wk diets containing 2.4 g/100 g of sunflower oil, fish oil, olive oil, lard or coconut oil. We measured alanyl-, arginyl-, cystinyl-, pyroglutamyl-, aspartyl- and glutamyl-specific aminopeptidase activities using arylamides as substrates. Serum total cholesterol levels were higher in mice fed diets containing saturated oils (lard and coconut) than in those consuming sunflower oil, which is unsaturated. Two of the serum aminopeptidase A activities (aspartyl and glutamyl aminopeptidase) increased progressively with the degree of saturation of the dietary fatty acids; activities were significantly greater in mice fed coconut oil than in those fed sunflower or fish oil. Therefore, the substrates hydrolyzed by this activity as well as their functions may be similarly affected. These results may have some implication for the treatment of cardiovascular disease.

Oleate, linoleate and cholesterol differently modify aspartyl- and glutamyl-aminopeptidase activities in primary cultures of rat astrocytes.

The intake of mono- and polyunsaturated fatty acids has been associated with a minor risk of cardiovascular diseases including hypertension. Changes in levels of fatty acids may also modify the cell activity and may be related with alterations in different regulatory processes. Aminopeptidases are zinc-metalloenzymes which metabolise circulating peptide hormones in several tissues. Glutamyl-aminopeptidase (GluAP) and to a lesser extent, aspartyl-aminopeptidase (AspAP), are related with angiotensin metabolism in the renin-angiotensin system. The present work was designed to study the effect of a range of concentrations (1-100 microM) of oleic and linoleic acids and cholesterol present in the culture medium on the activity of GluAP and AspAP in the culture of rat cerebral cortical astrocytes taken from 21-day-old fetuses. The results showed that oleic acid inhibits, while linoleic acid stimulates the activity of AspAP. Both fatty acids inhibit the activity of GluAP. Cholesterol stimulates the activity of both enzymes. On the basis of these results, a functional link may exit between the effects of fatty acids on hypertension and the modulation of aminopeptidase activity by these compounds in rat astrocytes, as an example of target cell type in the central nervous system.

[Aminoglycoside antibiotics neomycin and kanamycin inhibit the increase of of pyroglutamyl aminopeptidase activity by depolarizing synaptosomes of the frontal cortex of the rate]. / Los antibióticos aminoglucósidos neomicina y kanamicina inhiben el incremento de actividad piroglutamato aminopeptidasa obtenido tras despolarización de sinaptosomas de corteza frontal de rata.

INTRODUCTION: Thyrotrophin releasing hormone (TRH) has emerging in the last few years as a neuropeptide with important functions, not only as neurohormone into the hypothalamus-pituitary axis, but as neurotransmitter in several areas of the nervous system. Although little is known about its extra-endocrine functions, TRH has been related with several types of psychiatric disorders. Pyroglutamyl aminopeptidase (pGluAP) is the enzyme involved in the degradation of TRH. OBJECTIVES: The present research studies the levels of pGluAP activity under basal (resting) and KCl-stimulated (depolarized) conditions. The role of intracellular free calcium homeostasis, by means of the aminoglycoside antibiotics neomycin and kanamycin as voltage-dependent calcium channels blockers, is also studied. MATERIAL AND METHODS: Both pGluAP activity and intracellular free calcium concentration were analyzed in synaptosomes obtained from the frontal cortex of rats. Synaptosomes were incubated in artificial cerebrospinal fluid, under basal (resting) or KCl-stimulated (depolarized) conditions, with of without neomycin or kanamycin at different concentrations. RESULTS: Depolarization increases significantly pGluAP activity, which is completely abolished by neomycin and kanamycin at the lower concentrations used. On the contrary, aminoglycoside antibiotics do not block completely the increase on intracellular free calcium concentration induced by depolarization. Under basal conditions, no changes were found on pGluAP activity nor intracellular free calcium. CONCLUSIONS: pGluAP activity could regulate the neurotransmitter/neuromodulatory functions of TRH trough intracellular free calcium movements through aminoglycoside-sensitive voltage-dependent calcium channels. A role for inositol 4,5-bisphosphate breakdown products is also suggested.

Los antibióticos aminoglucósidos neomicina y kanamicina inhiben el incremento de actividad piroglutamato aminopeptidasa obtenido tras despolarización de sinaptosomas de corteza frontal de rata / Depolarization increases pyroglutamyl aminopeptidase activity in rat frontal cortex synaptosomes. Inhibitory effect of aminoglycoside antibiotics neomycin and kanamycin

Introducción. Uno de los neuropéptidos que está alcanzando un gran interés en los últimos años es la tirotropina o TRH, tanto por su papel como neurohormona en el eje hipotálamo-hipofisiario, como por su poco conocida función extra-endocrina. La acción neurotransmisora/neuromoduladora de la TRH es finalizada por la enzima piroglutamato aminopeptidasa (pGluAP). Objetivos. El presente trabajo se ha diseñado para estudiar el comportamiento de la actividad pGluAP en una situación basal o de reposo y tras la despolarización con KCl 50 mM. Para comprobar su posible relación con los niveles de calcio libre citosólico, se han utilizado los antibióticos aminoglucósidos neomicina y kanamicina como bloqueantes de canales de calcio dependientes de voltaje. Material y métodos. La actividad pGluAP, así como los niveles de calcio libre citosólico, se determinaron en sinaptosomas de corteza frontal de rata. Para ello se mantuvieron los sinaptosomas tanto en condiciones basales como despolarizantes, en un medio de incubación en presencia o ausencia de neomicina y kanamicina a diferentes concentraciones. Resultados. La despolarización de los sinaptosomas provoca un incremento altamente significativo de la actividad pGluAP, el cual es inhibido completamente por neomicina y kanamicina. Sin embargo, estos antibióticos aminoglucósidos no bloquean por completo el incremento en los niveles de calcio libre citosólico inducido por la despolarización. Ninguno de los parámetros estudiados se modifica en condiciones basales. Conclusiones. La actividad pGluAP puede controlar la función neurotransmisora/neuromoduladora de la TRH a través de los movimientos de calcio libre citosólico originados a través de canales de calcio sensibles a antibióticos aminoglucósidos, si bien hay que considerar el posible papel de los mensajeros intracelulares derivados del inositol 4,5-bisfosfato (AU)

[In vitro study of the effect of ethanol on pyroglutamyl aminopeptidase activity in mouse synaptosomes under basal and stimulated conditions]. / Estudio in vitro del efecto del etanol sobre la actividad piroglutamato aminopeptidasa en sinaptosomas de ratón en condiciones basales y despolarizantes.

INTRODUCTION AND OBJECTIVE: Pyroglutamyl aminopeptidase (pGluAP) is an omega peptidase which removes pyroglutamyl N-terminals residues from peptides and arylamidase derivatives. This activity is thought to be involved in the regulation of several physiological mechanisms on the central nervous system. pGluAP can modulate various susceptible endogenous substrates such as thyrotrophin-releasing hormone (TRH). It is well known that TRH plays an important role in the modulation of the behavioral changes induced by ethanol and others drugs. The aim of this work was to study the in vitro effects of ethanol (25, 50 and 100 mM) on the pGluAP activity and its ability for modulating the TRH. MATERIAL AND METHODS: pGluAP activity was measured in synaptosomes from cerebral cortex of mouse, using pyroglutamyl-beta-naphthylamide as substrate in basal and stimulated (K+ 25 mM) conditions, and in presence or absence of calcium on the buffer. RESULTS: In basal conditions, ethanol produced an inhibition of the pGluAP activity in presence or absence of calcium, being this inhibition non dose-related. However, the stimulation with K+ 25 mM did not produce a modification of pGluAP activity in presence of calcium, but produced a light increase in absence of it. Depolarization in presence or absence of calcium and ethanol produced an inhibition of pGluAP activity, which changed in function of the ethanol concentration used. CONCLUSIONS: Ethanol modifies pGluAP activity in basal conditions by a mechanism independent of calcium, but the changes observed after the stimulation with high K+ may be due to a calcium-dependent mechanism. These variations of pGluAP activity induced by ethanol, and their effects on their endogenous substrates, specially TRH, may be responsible for the behavioral changes associated to the alcoholism and mediated by TRH.

Estudio in vitro del efecto del etanol sobre la actividad piroglutamato aminopeptidasa en sinaptosomas de ratón en condiciones basales y despolarizantes / In vitro study on the effect of ethanol on pyroglutamyl aminopeptidase activity in mouse synaptosomes under basal and stimulated conditions

Introducción y objetivo. El enzima piroglutamato-aminopeptidasa (pGluAP) es una omega-peptidasa que libera residuos piroglutamilo N-terminales de péptidos y derivados arilamidas. Su actividad parece estar involucrada en la regulación de diferentes procesos fisiológicos en el sistema nervioso central mediante la modificación de distintos sustratos endógenos, en especial, la hormona liberadora de tirotropina (TRH). Puesto que se sabe que la TRH desempeña un papel importante en la modulación de los cambios de comportamiento inducidos por el alcohol etílico y otras drogas, el presente trabajo se ha diseñado para estudiar el efecto in vitro del alcohol etílico (25, 50 y 100 mM) sobre la actividad del enzima pGluAP y, por lo tanto, sobre su capacidad moduladora en dicho sustrato. Material y métodos. La actividad pGluAP se determinó en sinaptosomas obtenidos de corteza cerebral de ratón, utilizando piroglutamil-Beta-naftilamida como sustrato, tanto en condiciones basales como tras estimulación con K+ 25 mM, en un medio de incubación en presencia o ausencia de calcio. Resultados. En condiciones basales, el etanol inhibe la actividad pGluAP tanto en presencia como en ausencia de calcio en el medio; esta inhibición no se relaciona con la dosis. Sin embargo, la estimulación con K+ 25 mM no modifica la actividad pGluAP cuando el medio de incubación contiene calcio, pero la incrementa ligeramente en su ausencia. La despolarización en presencia o ausencia de calcio y etanol en el medio provoca, en general, una inhibición de la actividad pGluAP que varía en función de la dosis de etanol utilizada. Conclusiones. El etanol modifica la actividad pGluAP en condiciones basales por un mecanismo independiente del calcio, si bien las variaciones obtenidas tras la estimulación con K+ sí ocurren por un mecanismo dependiente del calcio. Estas modificaciones de la actividad de la pGluAP debidas al etanol y, por lo tanto, sus efectos sobre sus sustratos endógenos (especialmente la TRH) podrían ser responsables en parte de los cambios comportamentales asociados al alcoholismo y mediados por la TRH (AU)

Changes in membrane-bound leucine aminopeptidase activity during maturation and ageing of brain.

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 1-week-old 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.

[Subcellular distribution of leucine aminopeptidase during the development and aging of rat brains]. / Localización subcelular de leucina aminopeptidasa durante el desarrollo y envejecimiento cerebral de la rata.

INTRODUCTION: The aminopeptidases are considered to be enzymes which can regulate the activity of various neuropeptides. However, their precise function has not yet been fully determined. OBJECTIVE: With a view to studying the function of these enzymes at the cerebral level, the subcellular distribution of soluble leucine aminopeptidase from left and right cerebral hemisphere, in rats of different pre-natal and post-natal ages was determined as described in this paper. MATERIALS AND METHODS: The activity of soluble leucine aminopeptidase was determined fluorimetrically in the sub-cellular fractions obtained from the left and right cerebral hemispheres of rats of different pre-natal and post-natal ages: fetuses (of 19-21 days gestation), 1 week, 1, 5 and 24 months of age. RESULTS: No differences were observed between the cerebral hemispheres at any of the ages studied. However, significant differences were seen in the evolutionary behaviour of the two hemispheres. At all ages, except 24 months, there were significant differences between the fractions. In the fetuses, most activity was seen at the level of the synaptosomal raw fraction and least in the microsomal. At 1-week-old, the highest levels were seen at a microsomal level. In rats of 1 and 5 months of age, the highest levels were detected at synaptosomal level. With regard to the evolution with age, in the homogenate, nuclear fraction, cytosol and mitochondrial fraction a similar profile was observed, with a decrease after birth and an increase at one month, being stable at the other ages. At a synaptosomal level the profile is similar, although the differences are much more marked, with a drastic decrease in activity at two years of age. By contrast, the microsomal fraction showed a very different evolutionary profile with an increase after birth, becoming stable at later ages. CONCLUSIONS: The activity of soluble leucine aminopeptidase shows a heterogeneous subcellular distribution together with significant differences during cerebral development and ageing. The behaviour of this enzyme may reflect the functional state of its neuropeptide substrates.

Developmental changes of soluble and membrane-bound aspartate aminopeptidase activities in rat brain.

Specific soluble and membrane-bound aspartyl-naphthylamide hydrolyzing activities were assayed in brain subcellular fractions from rat fetuses (19-20 days of gestation), and from 1-week-old and 1-, 5- and 24-month-old rats. Both enzymatic activities showed a heterogeneous distribution, with highest concentrations mainly in the microsomal fraction. Membrane-bound activity was in most cases higher than soluble activity. With the exception of soluble activity in the nuclear and microsomal fractions, significant age-related changes were observed in all fractions for both enzymatic activities. Soluble activity showed a homogeneous developmental profile in most of the fractions, with the lowest levels in 1-month-old rats and the highest in 1-week and 5-month-old animals. However, changes in the microsomal fraction did not follow the pattern displayed by the rest of the fractions. No clear developmental profile in specific membrane-bound activity was observed, each fraction exhibiting a different sequence of changes. Whereas in 24-month-old-rats there was a significant increase in activity in homogenate, nuclear and microsomal fractions, a significant decrease was observed in the synaptosomal fraction. These results may reflect the functional status of the endogenous substrates of the enzymes.

Subcellular distribution of soluble and membrane-bound Arg-beta-naphthylamide hydrolyzing activities in the developing and aged rat brain.

The subcellular distribution of soluble and membrane-bound Arg-beta-naphthylamide-hydrolyzing activities was studied in the left and right rat brain during development and aging. During development, the soluble activity was heterogeneous, whereas adult animals showed the highest activity in the synaptosomal fraction. However, except in fetuses, membrane-bound activity was greatest in the microsomal fraction. Except in microsomal and myelin fractions, soluble and membrane-bound activities showed a decrease in 1-wk-old rats compared with fetuses and a subsequent increase to adult levels in 1-mo-old rats. This profile differed in the microsomal fraction, which increased steadily throughout development. In the synaptosomal fraction, both activities were lower in 24-mo-old rats than in 5-mo-old animals. No differences between the hemispheres were observed in soluble or membrane-bound fractions at any age tested.

Developmental and ageing changes in aminopeptidase activities in selected tissues of the rat.

Aminopeptidase activities, assayed as arylamidase activities, were investigated in selected tissues of 1, 6, 12 and 24-month-old rats. The enzyme activities were found to have a heterogeneous distribution and age-related changes were observed. The highest levels of soluble arginyl-aminopeptidase activity were detected in brain homogenate at all the studied ages, whereas membrane-bound activity presented the highest levels in brain and kidney in the four ages tested. Aspartyl-aminopeptidase activity was detected mainly in the particulate fraction of kidney at all four ages. In 1, 6 and 12-month-old animals, soluble aspartyl-aminopeptidase activity was also higher in the kidney than in the rest of the tissues, whereas in the group of 2-year-old rats, the highest levels were found in both kidney and liver. Age-related changes were observed in all the studied tissues and for all the assayed enzymatic activities. In general, the maximal levels were detected in both the youngest and the oldest animals, and the minimal ones in 6 and 12-month-old rats. However, in the adrenals, the soluble and membrane-bound arginyl-aminopeptidase activity was higher in 6-month and 2-year-old rats than in 1-month and 12-month-old rats. These changes may reflect the functional status of the susceptible endogenous substrates of aminopeptidases.

Daily rhythm of aspartate aminopeptidase activity in the retina, pineal gland and occipital cortex of the rat.

The levels of specific soluble aspartyl aminopeptidase activity were assayed in retina, occipital cortex, anterior pituitary, posterior pituitary, pineal gland and serum of adult male rats, using Asp-2-naphthylamide as substrate, in a 12:12 h light:dark cycle (7-19 h light). Significant diurnal variations appeared in retina, pineal gland, occipital cortex and serum. In addition, different patterns of diurnal variation of the enzymatic activity were observed in the tissues analyzed. A regular increase of the activity was noticed at the end of the dark period in all the tissues as a common feature, except in serum, in which the enzymatic activity reached a peak in the middle of the light period, decreasing progressively during the dark hours. After the last hours of darkness, the pattern of variation in the activity differed in each tissue. These diurnal variations in aspartyl aminopeptidase activity could reflect the functional status of its putative endogenous substrates, such as angiotensin II, and it may also suggest the existence of differential regulatory mechanisms associated with each location.

Bilateral distribution of aminopeptidase activities in selected structures of a photoneuroendocrine circuit and other rat brain areas.

Provided that soluble aminopeptidases, the most abundant proteolytic enzymes found in brain, are involved in the metabolism of several neuropeptides, their activity could be a reflect of neuropeptide function. Therefore, in order to analyze their rate of participation, we have measured 4 soluble aminopeptidase activities: leucine aminopeptidase, arginine aminopeptidase, aspartate aminopeptidase, and pyroglutamate aminopeptidase, using arylamide derivatives as substrates, in selected structures integrating the photoneuroendocrine circuit related to the melatonin rhythm generating system and other rat brain areas. The regional distribution of all the activities was heterogenous: a 3-fold (leucine-, arginine- and aspartate-aminopeptidase) and 5-fold (pyroglutamate aminopeptidase) difference was observed between the regions with the highest and lowest activity. Significant differences were displayed between the left and right retina for pyroglutamate aminopeptidase and arginine aminopeptidase activities. High levels of pyroglutamate aminopeptidase were evident in the retina and adenohypophysis, which is consistent with a role for thyrotropin releasing hormone in photoreceptive mechanisms, and support its well established role in controlling thyrotropin releasing hormone in anterior pituitary. The presence of a high activity rate of aspartate aminopeptidase in adenohypophysis implies an active participation of angiotensin peptides at this level.