Subcellular distribution of pyroglutamyl-peptidase I activity in the developing rat cerebellum.

In this work we analyzed the activity of pyroglutamyl (pGlu)-peptidase I in several subcellular fractions of the rat cerebellum during its development. The aim of this study is to determine if there is a developmental redistribution of this enzyme activity and if this fact could indicate changes in the role of the enzyme as age progresses. Results show that pGlu-peptidase I is widely distributed, but not homogeneously, in all the subcellular fractions that were studied, in both soluble and particulate forms. Significantly the distribution of the enzyme changes with development. Thus, in the soluble synaptosomal fraction, the pGlu-peptidase I activity is low until PD9 and the activity increases significantly from PD9 to PD15, when it reaches adult levels. In contrast, in the cytosolic fraction, the pGlu-peptidase I activity is high from fetal day 22 to postnatal day 6, and then decreases significantly until postnatal day 90.

Subcellular ontogeny of brain pyroglutamyl peptidase I.

The present work studies pyroglutamyl-peptidase I activity in several subcellular fractions of the developing brain. In the synaptosomal fraction, soluble pGlu-peptidase I activity is low until postnatal Day 9 (with a peak at postnatal Day 2) and the activity increases at postnatal Day 15. In later stages there are not significant changes of synaptosomal pGlu-peptidase I activity. However, in the cytosolic fraction the activity is high from ED22 until postnatal Day 9, and afterwards decreases. The changes in the particulate fractions are generally less drastic.

Lithium alters mu-opioid receptor expression in the rat brain.

Lithium can potentiate the effects of antidepressant drugs and alters morphine analgesia and phosphoinositide turnover. Analysis of mu-opioid receptor immunostaining after chronic lithium administration in rats revealed an increase in the density of cells expressing mu-opioid receptors in the caudatus-putamen, the dentate gyrus, the lateral septum and the frontal, parietal and piriform cortices. These data suggest that mu-opioid receptor expression in the rat forebrain is altered by in vivo chronic lithium treatment. This could be a compensatory mechanism, induced in part by the effects of lithium on mu-opioid receptor transduction mechanism.

Ontogeny of puromycin-sensitive and insensitive aminopeptidase activities in several subcellular fractions of the rat brain.

Puromycin-sensitive and insensitive aminopeptidase (aminopeptidase M) activities are measured in several subcellular fractions of the rat brain cortex and subcortex during the first postnatal month. Tyr-beta-naphthylamide has been used as substrate and 20 microM puromycin as selective inhibitor. We have found that puromycin-sensitive aminopeptidase activity increases twofold in the synaptosomal and mitochondrial fractions in the first 6-9 postnatal days, just during the period of axonal and dendritic growth. This enzyme also has significant age-related changes in the nuclear fraction. The developmental pattern is different, depending on the subcellular fraction analyzed. Significant developmental changes of puromycin-insensitive aminopeptidase (aminopeptidase M) are only found in the myelinic and microsomal fractions and they are less significant than those found in the puromycin-sensitive aminopeptidase. It has been suggested that these enzyme activities could be involved in processes of cell proliferation, differentiation, and maturation.

Effect of lithium treatments on the tyrosine-aminopeptidase activities in the rat brain and the pituitary gland.

Lithium (CAS 7439-93-2) is being utilized widely for the treatment of manic-depressive illness. However, the neurochemical mechanism of the ion action still remains relatively obscure. In the present work, the effect of acute lithium administration on soluble and membrane-bound enkephalin-degrading tyrosine-aminopeptidase activities in discrete brain areas and the pituitary gland is described. Tyrosine-aminopeptidase activities have been measured fluorometrically using Tyr-beta-naphthylamide as substrate and puromycin as selective inhibitor of one of the two membrane activities. Soluble and membrane-bound puromycin-insensitive Tyr-aminopeptidase activities are greater in the pituitary gland after treatment with 5 and 10 meq/kg of lithium. It is suggested that these enzyme activities could play a part in the lithium action mechanism.

Effects of fluoxetine administration on mu-opoid receptor immunostaining in the rat forebrain.

Fluoxetine is a selective serotonin reuptake inhibitor. Analysis of mu-opioid receptor immunostaining after chronic fluoxetine administration in rats revealed an increase in the density of cells expressing mu-opioid receptors in the caudatus-putamen, the dentate gyrus, the lateral septum and the frontal, parietal and piriform cortices. These data suggest that mu-opioid receptor expression in the rat forebrain is altered by in vivo chronic fluoxetine treatment.

Effect of imipramine on enkephalin-degrading peptidases.

In recent years, there has been increasing evidence of the involvement of the endogenous opioid system in mental depression and its treatment. In this work, we have measured the effect of imipramine on enkephalin-degrading peptidases in several rat brain areas. Aminopeptidase activities have been assayed using Tyr-beta-naphthylamide as substrate and puromycin as selective inhibitor. Dansyl-D-Ala-Gly-Phe(pNO2)-Gly has been the substrate for neutral endopeptidase 24.11. Imipramine in vitro inhibits puromycin-sensitive activities in all brain areas studied, without affecting the rest of the enzymes assayed. However, subacute imipramine treatment increases neutral endopeptidase activity in the hypothalamus and chronic treatment increases this activity in the hypothalamus and the striatum. These results suggest to us that enkephalin-degrading peptidases are involved in the acute and chronic action mechanism of imipramine and reinforce the idea that the central enkephalinergic activity is dynamically changed during the treatment of depressive illness.

Infraorbital nerve transection increases NADPH-diaphorase activity in the rat mesencephalic trigeminal nucleus.

Complete information concerning possible alterations in nitric oxide production in the rat brainstem trigeminal system after peripheral nerve lesion is still lacking. This being the case, density of NADPH-diaphorase positive neurons in the trigeminal mesencephalic nucleus after experimental infraorbital nerve transection was studied. In the lesioned side, an ipsilateral increase in NADPH-d positive neurons was found at postoperative days 4 and 6 with respect to contralateral, without changes after a 2 months period. These data suggest that nitric oxide could be involved in regeneration of afferent fibers concerned with the periodontal receptors of maxillary teeth.

Effects of imipramine administration on mu-opioid receptor immunostaining in the rat forebrain.

Imipramine hydrochloride (CAS 113-52-0) is a widely used antidepressant and can interact with opioid receptors. However, complete information concerning possible regional alterations in mu-opioid receptor expression in the rat forebrain after chronic treatment with this substance is still lacking. This being the case, analysis of mu-opioid receptor immunostaining in several regions of the rat brain after imipramine administration in vivo was made, and an increase in the density of cells expressing mu-opioid receptors in the caudatus-putamen, the dentate gyrus and the frontal, parietal and piriform cortices after chronic imipramine treatment, with respect to controls, was found. These data suggest that mu-opioid receptor expression in the rat forebrain is altered by in vivo chronic imipramine treatment.

Toluene alters brainstem enkephalinergic system in rats.

Acute exposure to high doses of toluene can generate respiratory depression. However, neurotoxic mechanism of its action in the brainstem is not completely clear. In this work, acute, but not subchronic, exposure of rats to toluene increased leu-enkephalin immunostaining in several myelencephalic nuclei implicated in cardiorespiratory control. Due to the physiological role of enkephalins in the central regulation of breathing, it is suggested that the enkephalinergic system could play a role in neurotoxic respiratory depression induced by high dose acute toluene exposure.

Polyploidization and exit from cell cycle as mechanisms of cultured melanoma cell resistance to methotrexate.

Numerous malignant neoplasias are found to contain varying proportions of high-ploidy cells. Although the role they play in the tumor is poorly understood, several lines of evidence suggest that these cells could be especially resistant to various aggressions, a possibility of great interest in cancer treatment. In the present study, we tested this hypothesis through the analysis of the presence of high-ploidy cells following the administration of the chemotherapeutic agent methotrexate. We also determined the expression of two proliferation markers, PCNA and CDK1, after methotrexate-treatment. Cultured cells from the murine melanoma B16F10 were treated with high doses of methotrexate for seven days prior to determination of DNA content and proliferation markers. Our results showed an obvious increase in the mean ploidy of this population. Specifically, there was a dramatic reduction in the proportion of tetraploid cells (predominant in the original population), and an increase in the proportion of cells with higher ploidies, particularly those whose DNA content was greater than 8c, including some cells with ploidies greater than 16c. Furthermore, there was a reduction in the number of PCNA-expressing cells and the reduction was much more marked in the case of CDK1 that was almost absent in the modal-ploidy treated cells. These alterations concerning ploidy and expression of proliferation markers had completely reverted two weeks after withdrawal of the drug. Our results indicate that methotrexate at a high dosage selects a cell population heterogeneous concerning its ploidy level, composed of one subpopulation of high-ploidy cells and another of modal-ploidy cells that, considering its lack of CDK1 expression, would remain in a latent state to evade the effects of the drug.

Effects of acute lidocaine administration on the rat prosencephalic enkephalinergic system.

Lidocaine is a local anesthetic widely used in therapeutics and as antiarrhythmic agent. However, information concerning possible alterations in the enkephalinergic system after acute treatment with this substance is not complete. This being the case, we focused on analyzing enkephalin immunostaining in several regions of the rat brain after lidocaine administration. We could not find significative changes in rat prosencephalon. These data might suggest that the enkephalinergic system is not altered by lidocaine.

Soluble and membrane-bound pyroglutamyl-peptidase I activity in the developing cerebellum and brain cortex.

Developmental changes of soluble and particulate pyroglutamyl (pGlu)-peptidase I activities in the rat brain cortex and in the cerebellum are described in this work. The enzyme activity has been measured spectrofluorimetrically using pGlu-b-naphthylamide as substrate (in the presence and absence of EDTA and DTT, necessary activators of the enzyme) in both soluble and particulate fractions. In the soluble fraction of the cerebellum and brain cortex, pGlu-peptidase I activity is high in the perinatal period and decreases two or three folds subsequently, at a later stage in the cerebellum than in the brain cortex, reaching adult levels at the end of the first postnatal month. The decrease in the activity of pGlu-peptidase observed in this work coincides with increasing levels of brain thyroliberin concentration after the second postnatal week. The particulate pGlu-peptidase I activity, obtained after osmotic shock and high-salt treatment, shows less significant changes during brain development in the areas under study. It is suggested that cytosolic pGlu-peptidase I could play a part in the normal development of the rat central nervous system.

Effect of lidocaine administration on the enkephalin-degrading aminopeptidase activities in discrete areas of the rat brain.

1. The levels of three aminopeptidase activities involved in the degradation of enkephalins (soluble, M and MII) in several rat brain areas were studied after lidocaine administration. 2. Soluble aminopeptidase activity showed decreases in the frontal cortex and in the pituitary gland after treatment. 3. No significant changes in the complete membrane-bound aminopeptidase activity were appreciated in any other of the brain areas. 4. However, decreases of the membrane-bound puromycin-insensitive aminopeptidase activity in the frontal cortex, the hippocampus and the thalamus, after lidocaine administration, were observed.

Subcellular analysis of Tyr-aminopeptidase activities in the developing rat cerebellum.

The endogenous opioid system seems to play important roles in the developing cerebellum. The first opioid peptide isolated, Met-enkephalin, is expressed transiently in this brain area. In the present study, several enzyme activities capable of hydrolyzing enkephalins are measured during the first month of cerebellar development, using Tyr-beta-naphthylamyde as substrate and puromycin as inhibitor of one of the membrane-bound aminopeptidases. Puromycin-sensitive soluble and membrane-bound aminopeptidase activities decrease in the synaptosomal and mitochondrial fractions at the end of the first month of life, just when enkephalin-like immunoreactivity decreases in the cerebellum. Membrane-bound enzyme also decreases in the myelinic fraction. Synaptosomal activity increases after birth, coinciding with decreases in the activity in the microsomal fraction. Puromycin-insensitive and membrane-bound aminopeptidase shows less significant developmental changes and they occur mainly in the first week of life, coinciding with the axonal and dendrite growth. These results could suggest a possible role of these enzymes, together with the rest of the opioid system, in cerebellar development.

Interaction mechanisms of imipramine and desipramine with enkephalin-degrading aminopeptidases in vitro.

In the last few years, considerable evidence has appeared concerning the importance of the opioid systems in the action mechanism of some antidepressant drugs. This action mechanism could be mediated through the inhibition of the enzymes responsible for enkephalin degradation. In this sense, imipramine treatment in vivo increases the enkephalin levels, and this effect is enhanced by inhibitors of enkephalin-degrading enzymes. The present work shows the effects in vitro of imipramine and its active metabolite desipramine on the activities of two membrane-bound enkephalin-degrading aminopeptidases present in rat brain. Imipramine and desipramine in vitro do not affect the aminopeptidase M activity, but they reversibly inhibits the aminoeptidase MII. The enzyme kinetic analysis shows that this enzyme molecule has two different binding sites for each drug, which exert a mixed type enzyme inhibition.

Genesis and evolution of high-ploidy tumour cells evaluated by means of the proliferation markers p34(cdc2), cyclin B1, PCNA and 3[H]-thymidine.

Although cell polyploidization is not an infrequent event in mammalian cells and is common in tumours, the mechanisms involved are not well understood. Using the murine B16 cell line as a model, we evaluated the role of some key proteins involved in cell cycle progression: p34(cdc2), cyclin B1 and PCNA. By means of flow cytometry, we showed that both in modal- and in high-ploidy subpopulations, almost all cells were p34(cdc2)-positive. In the modal-ploidy subpopulation only 17.1% cells were cyclin B1-positive and 85.6% PCNA-positive; in contrast, in the high-ploidy subpopulation up to 91.8% cells were cyclin B1-positive and 97.3% cells were PCNA-positive (P < 0.001). Immunofluorescence microscopy showed that PCNA was located in the nucleus; p34(cdc2), both in the nucleus and cytoplasm; and cyclin B1 yielded a cytoplasmic spotted pattern with a perinuclear reinforcement. After a 24-h incubation with 3[H]-thymidine followed by withdrawal of the isotope, high-ploidy cells remained labelled 8 days after thymidine withdrawal, in contrast to modal-ploidy cells. Taken together, our results suggest that polyploid cells are not quiescent, their cell cycle is longer than that of the modal-ploidy population, and they maintain cyclin B1 throughout the cycle, which may contribute to their genesis by impeding the exit from mitosis.

Membrane-bound tyrosine aminopeptidase activities in the rat brain throughout the estrous cycle.

Puromycin sensitive and insensitive membrane-bound aminopeptidase activity levels during the estrous cycle in several brain areas have been described in this research. We have found the highest aminopeptidase M activity levels during the proetrous stage in the hypothalamus, the amygdala and the pituitary gland. Since this enzyme has been involved in opioid peptide metabolism, it is suggested that aminopeptidase M could play a part in the decrease in the inhibitory influence of the endogenous opioids peptides that participate in the LH surge.

Age-related changes in the soluble and the membrane-bound Tyr-aminopeptidase activities in several areas of the male and female rat brain.

It has been recently suggested that enkephalins might play a normal role in the regulation of cellular development in brain. Since the major pathway of enkephalin degradation seems to occur under the action of aminopeptidases, in the present paper we describe the changes in Tyr-aminopeptidase activities during several stages of the rat (male and female) brain development (9, 12, 15, 20 and 25 days postbirth). The enzyme activities (soluble and membrane-bound) were detected using Tyr-2-naphthylamide as substrate. No sexual differences were observed. However, significant rises from the 9th to the 15th postnatal day in the soluble peptidase activity were appreciated. Aminopeptidase M shows decreases in the activity with age. It is suggested that not only the enkephalins but also the enkephalin-degrading enzymes could play a part in the maturation of the rat brain.